Nucleotide derivatives and methods of use thereof

ABSTRACT

Disclosed herein, inter alia, are compounds, compositions, and methods of thereof in the sequencing a nucleic acid.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a § 371 national stage of PCT InternationalApplication No. PCT/US2017/033,939, filed May 23, 2017, and claims thebenefit of U.S. Provisional Applications Nos. 62/477,945, filed Mar. 28,2017; 62/365,321, filed Jul. 21, 2016; and 62/340,419, filed May 23,2016, each of which is incorporated herein by reference in its entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII FILE

This application incorporates-by reference nucleotide and/or amino acidsequences which are present in the file named“181121_88662-A-PCT-US_Sequence_Listing_CAS.txt”, which is 5.61kilobytes in size, and which was created Nov. 20, 2018 in the IBM-PCmachine format, having an operating system capacity with MS-Windows,which is contained in the text file being filed Nov. 21, 2018 as part ofthis application.

BACKGROUND

DNA sequencing is a fundamental tool in biological and medical research;it is an essential technology for the paradigm of personalized precisionmedicine. Among various new DNA sequencing methods, sequencing bysynthesis (SBS) is the leading method for realizing the goal of the$1,000 genome. Currently, the widely used high-throughput SBS technology(Bentley D R. et al. Nature, 2008, 456, 53-59) determines DNA sequencesduring the polymerase reaction using cleavable fluorescently labelednucleotide reversible terminator (NRT) sequencing chemistry that hasbeen previously developed (Ju J et al. 2003, U.S. Pat. No. 6,664,079; JuJ et al. Proc Natl Acad Sci USA, 2006, 103, 19635-19640). Thesecleavable fluorescent NRTs were designed based on the rationale thateach of the nucleotides is modified by attaching a unique cleavablefluorophore to the specific location of the base and capping the 3′-OHgroup with a small reversible-blocking moiety so they are stillrecognized by DNA polymerase as substrates. A disadvantage of theabovementioned SBS approach is the production of a small molecular“scar” (e.g., a propargylamine or a modified propargylamino moiety) atthe nucleotide base after cleavage of the fluorescent dye from theincorporated nucleotide in the polymerase reaction. The growing DNAchain accumulates these scars through each successive round of SBS. Atsome point, the residual scars may be significant enough to interferewith the DNA double helix structure, thereby negatively affecting DNApolymerase recognition and consequently limiting the read length.Accumulated research efforts indicated that the major challenge for thisapproach is that DNA polymerase has difficulty accepting 3′-Obulky-dye-modified nucleotides as substrates, because the 3′ position onthe deoxyribose of the nucleotides is very close to the amino acidresidues in the active site of the DNA polymerase while in the ternarycomplex formed by the polymerase with the complementary nucleotide andthe primed template. Accordingly, there is a need for the use inscarless SBS, and synthesis of, 3′-O modified nucleotides andnucleosides that are effectively recognized as substrates by DNApolymerases, are efficiently and accurately incorporated into growingDNA chains during SBS, have a 3′-O blocking group that is cleavableunder mild conditions wherein cleavage results in a 3′-OH, and permitlong SBS read-lengths. Disclosed herein, inter alia, are solutions tothese and other problems in the art.

BRIEF SUMMARY OF THE INVENTION

In an aspect is provided a nucleotide analogue having the formula:

B is a base or analogue thereof. L¹ is covalent linker. L² is covalentlinker. L⁴ is covalent linker. X is a bond, O, NR^(6A), or S. R³ is —OH,monophosphate, polyphosphate or a nucleic acid. R^(4A) and R^(6A) areindependently hydrogen, —OH, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂,—CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁵ is a detectable label, anchor moiety, oraffinity anchor moiety. R⁶ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R^(4B) is hydrogen or —OR^(7A), wherein R^(7A)is hydrogen or a polymerase-compatible moiety. R¹² is a complementaryaffinity anchor moiety binder. R¹³ is a detectable label. The symbol“---” is a non-covalent bond.

In an aspect is provided a thermophilic nucleic acid polymerase complex,wherein the thermophilic nucleic acid polymerase is bound to anucleotide analogue having the formula:

B is a base or analogue thereof. L¹ is covalent linker. L² is covalentlinker. L⁴ is covalent linker. R³ is —OH, monophosphate, polyphosphateor a nucleic acid. R^(4A) and R^(6A) are independently is hydrogen, —OH,—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl,—CH₂Br, —CH₂I, —CN, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R^(4B)is hydrogen, —OH, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂,—CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —X—R⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. X is a bond, O, NR^(6A), or S. R⁵ is adetectable label, anchor moiety, or affinity anchor moiety. R⁶ ishydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F,—CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁷ ishydrogen or —OR^(7A), wherein R^(7A) is hydrogen or apolymerase-compatible moiety. R¹² is a complementary affinity anchormoiety binder. R¹³ is a detectable label. The symbol “----” is anon-covalent bond.

In another aspect is provided a thermophilic nucleic acid polymerasecomplex (e.g., 9°N nucleic acid polymerase complex), wherein the nucleicacid polymerase (e.g., thermophilic) is bound to a nucleotide analogue,wherein the nucleotide analogue includes a fluorescent dye with amolecular weight of at least about 140 Daltons, and wherein thefluorescent dye is covalently bound at the 3′ position of the nucleotideanalogue.

In an aspect is provided a method of incorporating a nucleotide analogueinto a nucleic acid sequence including combining a thermophilic nucleicacid polymerase, a primer hybridized to nucleic acid template, and anucleotide analogue including a detectable label, within a reactionvessel and allowing the thermophilic nucleic acid polymerase toincorporate the nucleotide analogue into the primer therebyincorporating a nucleotide analogue into a nucleic acid sequence.

In an aspect is provided a method for sequencing a nucleic acid,including: (i) incorporating in series with a thermophilic nucleic acidpolymerase, within a reaction vessel, one of four different labelednucleotide analogues into a primer to create an extension strand,wherein the primer is hybridized to the nucleic acid and wherein each ofthe four different labeled nucleotide analogues include a uniquedetectable label; (ii) detecting the unique detectable label of eachincorporated nucleotide analogue, so as to thereby identify eachincorporated nucleotide analogue in the extension strand, therebysequencing the nucleic acid; wherein each of the four different labelednucleotide analogues are of the structure formula:

wherein the first of the four different labeled nucleotide analogues, Bis a thymine or uracil hybridizing base; in the second of the fourdifferent labeled nucleotide analogues, B is an adenine hybridizingbase; in the third of the four different labeled nucleotide analogues. Bis an guanine hybridizing base; and in the fourth of the four differentlabeled nucleotide analogues, B is an cytosine hybridizing base. B is abase or analogue thereof. L¹ is covalent linker. L² is covalent linker.L⁴ is covalent linker. X is a bond, O, NR^(6A), or S. R³ is —OH,monophosphate, polyphosphate or a nucleic acid. R^(4A) and R^(6A) areindependently hydrogen, —OH, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂,—CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁵ is a detectable label, anchor moiety, oraffinity anchor moiety. R⁶ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁷ is hydrogen or —OR^(7A), wherein R^(7A) ishydrogen or a polymerase-compatible moiety. R¹² is a complementaryaffinity anchor moiety binder. R¹³ is a detectable label. The symbol“----” is a non-covalent bond.

In another aspect is provided a method of incorporating a nucleotideanalogue into a nucleic acid sequence including combining a thermophilicnucleic acid polymerase, a primer hybridized to nucleic acid template,and a nucleotide analogue, within a reaction vessel and allowing thethermophilic nucleic acid polymerase to incorporate the nucleotideanalogue into the primer thereby incorporating a nucleotide analogueinto a nucleic acid sequence, wherein the nucleotide analogue includes afluorescent dye with a molecular weight of at least about 140 Daltons,and wherein the fluorescent dye is covalently bound at the 3′ positionof the nucleotide analogue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Scarless SBS using 3′-O-“anchor”-SS(DTM)-dNTPs and correspondinglabeled binding molecules (where “DTM” refers to the Dithiomethylgroup). (STEP 1) Addition of a DNA polymerase to the primed templatemoiety (only the primer strand is shown above) leads to theincorporation of a complementary 3′-O-“anchor”-SS(DTM)-dNTP to the 3′end of a primer with high efficiency and specificity. (STEP 2) Additionof labeled binding molecules to the corresponding primer extensionproduct leads to orthogonal binding of the labeled binding moleculeswith the corresponding “anchor” moiety in the 3′ end of the primerextension product; after washing away the unbound labeled molecule, thedetection of the unique label attached to the 3′ end of the primerextension product determines the identity of the incorporatednucleotide. (STEP 3) Addition of TCEP or THP results in the cleavage ofthe disulfide bond, and therefore to the removal of the label on theprimer extension product and the regeneration of the 3′-OH on the primerextension product. The repetition of STEP 1 through STEP 3 allows forcontinuous DNA sequence determination. The “Anchor” moiety and thelabeled binding molecule include any specifically reactive pair that canform a covalent bond or a stable noncovalent bond. The label can be afluorescent molecule, a FRET cassette or a fluorescent dendrimers.

FIGS. 2A-2E. Structures of 3′-O-Biotin-DTM-dNTPs(3′-O-Biotin-t-Butyldithiomethyl-dATP,3′-O-Biotin-t-Butyldithiomethyl-dCTP,3′-O-Biotin-t-Butyldithiomethyl-dGTP,3′-O-Biotin-t-Butyldithiomethyl-dTTP) and with Cy5 dye labeledstreptavidin as an example (wherein “DTM” refers to the Dithiomethylgroup). FIG. 2A: Cy5 Labeled Streptavidin. FIG. 2B:3′-O-Biotin-t-Butyldithiomethyl-dATP. FIG. 2C:3′-O-Biotin-t-Butyldithiomethyl-dCTP. FIG. 2D:3′-O-Biotin-t-Butyldithiomethyl-dGTP. FIG. 2E:3′-O-Biotin-t-Butyldithiomethyl-dTTP

FIGS. 3A-3B. Scarless one-color SBS using 3′-O-Biotin-SS(DTM)-dNTPs andCy5 labeled streptavidin. DNA polymerase incorporation reaction isconducted by using one of the four 3′-O-Biotin-SS-dNTPs, followed by theaddition of the Cy5 labeled streptavidin and imaging to determine DNAsequences as described in STEP 1 through STEP 4 (as shown in as 3.1 andrepeated in 3.2, 3.3 and 3.4). Each step consists of three parts: (PARTa) Add polymerase and one of the four 3′-O-Biotin-SS-dNTPs followed bywashing; if the added nucleotide is complementary to the nucleotide onthe template immediately next to the 3′ end of the primer, then theadded nucleotide will incorporate into the primer to produce a DNAextension product that has a Biotin at the 3′ end. (PART b) Add Cy5labeled streptavidin, which will bond to the Biotin at the 3′ end of theDNA extension product. (PART c) After washing away the unbound Cy5labeled streptavidin, perform imaging to detect the Cy5 signal for theidentification of the incorporated nucleotide. Following STEP 4,addition of THP to the DNA extension products will cleave the disulfidebond and regenerate a free 3′-OH group on the 3′ end of the DNAextension products. Sequentially repeat the process, consisting of STEP1 through STEP 4, followed by THP cleavage, for continuing sequencedetermination. The text over the arrows is as follows: 3.1: 1. (a) Add3′-O-Biotin-SS-dATP and DNA polymerase; (b) add Cy5-streptavidin; (c)imaging; 2. (a) Add 3′-O-Biotin-SS-dTTP and DNA polymerase; (b) addCy5-streptavidin; (c) imaging; 3. (a) Add 3′-O-Biotin-SS-dGTP and DNApolymerase; (b) add Cy5 labeled streptavidin; (c) imaging; 4. (a) Add3′-O-Biotin-SS-dCTP and DNA polymerase; (b) add Cy5 labeledstreptavidin; (c) imaging. 3.2; Repeat steps 1, 2, 3 and 4. 3.3: Repeatsteps 1, 2, 3 and 4. 3.4: Repeat steps 1, 2, 3 and 4.

FIG. 4. Structures of 3′-O-“Anchor”-SS(DTM)-dNTPs(3′-O-TCO-t-Butyldithiomethyl-dATP, 3′-O-PBA-t-Butyldithiomethyl-dCTP,3′-O-Biotin-t-Butyldithiomethyl-dGTP,3′-O-Azido-t-Butyldithiomethyl-dTTP). In this set of nucleotideanalogues, four different “anchor” moieties, TCO, PBA, Biotin and Azidogroups, are attached to the 3′-O of dATP, dCTP, dGTP and dTTP,respectively, through the DTM linkage, as shown in this figure.

FIG. 5. Structures of four-color labeled orthogonal binding molecules(Rox-Labeled Tetrazine, Alexa488-Labeled SHA, Cy5-Labeled Streptavidin,and R6G-Labeled Dibenzocyclooctyne) that bond specifically with the four“anchor” moieties in the nucleotide analogues(3′-O-TCO-t-Butyldithiomethyl-dATP, 3′-O-PBA-t-Butyldithiomethyl-dCTP,3′-O-Biotin-t-Butyldithiomethyl-dGTP,3′-O-Azido-t-Butyldithiomethyl-dTTP) listed in FIG. 4, as follows: Roxis attached to the Tetrazine (which specifically reacts with TCO);Alexa488 is attached to the SHA (which forms a stable complex with PBA);Cy5 is attached to the Streptavidin (which forms a stable complex withBiotin); and R6G is attached to the Dibenzocyclooctyne (DBCO, whichquickly forms a Triazole moiety with an N₃ group). Thus, each nucleotideanalogue listed in FIG. 4 can be labeled by a unique fluorescent dye.

FIGS. 6A-6D. Conjugates or complexes between DNA products produced byincorporating 3′-O “anchor” labeled nucleotides(3′-O-TCO-t-Butyldithiomethyl-dATP, 3′-O-PBA-t-Butyldithiomethyl-dCTP,3′-O-Biotin-t-Butyldithiomethyl-dGTP,3′-O-Azido-t-Butyldithiomethyl-dTTP) with four correspondingly-matchedlabeled binding molecules (Rox-Labeled Tetraine, Alexa488-Labeled SHA,Cy5-Labeled Streptavidin, and R6G-Labeled Dibenzocyclooctyne). Thereaction of the DNA extension product containing four “anchor” moietiesat the 3′-end with four correspondingly-matched labeled bindingmolecules leads to each incorporated nucleotide in the DNA extensionproduct being labeled with a unique dye. Thus, Rox will be tethered tothe 3′-end of a DNA extension product through a specific Tetrazine TCOligation to form PRODUCT 1; Alexa488 will be tethered to the 3′-end of aDNA extension product through a stable PBA-SHA complex to form PRODUCT2; Cy5 will be tethered to the 3′-end of a DNA extension product througha Biotin Streptavidin complex to form PRODUCT 3; and R6G will betethered to the 3′-end of a DNA extension product through triazoleformation via a click reaction between Dibenzocyclooctyne and an azidogroup to form PRODUCT 4.

FIG. 7. Scarless SBS using 3′-O-“anchor”-SS(DTM)-dNTPs(3′-O-TCO-t-Butyldithiomethyl(SS)-dATP,3′-O-PBA-t-Butyldithiomethyl(SS)-dCTP,3′-O-Biotin-t-Butyldithiomethyl(SS)-dGTP,3′-O-Azido-t-Butyldithiomethyl(SS)-dTTP) and fourcorrespondingly-matched dye labeled binding molecules (Rox-LabeledTetrazine, Alexa488-Labeled SHA, Cy5-Labeled Streptavidin, andR6G-Labeled Dibenzocyclooctyne). Addition of the DNA polymerase and thefour 3′-O-“anchor”-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP, 3′-O-PBA-SS-dCTP,3′-O-Biotin-SS-dGTP and 3′-O—N₃-SS-dTTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand to terminate DNA synthesis. Afterwashing away the unincorporated nucleotide analogues, add the dyelabeled binding molecules, which will specifically connect with each ofthe four unique “anchor” moieties at the 3′-end of each DNA extensionproduct to enable the labeling of each DNA product terminated with eachof the four nucleotide analogues (A, C, G, T) with the four distinctfluorescent dyes. Detection of the unique fluorescence signal from eachof the fluorescent dyes on the DNA products allows for theidentification of the incorporated nucleotide. Next, treatment of theDNA products with THP cleaves the SS linker, leading to the removal ofthe fluorescent dye and the regeneration of a free 3′-OH group on theDNA extension product, which is ready for next cycle of DNA sequencingreaction (as shown in the subsequent steps of FIG. 7). The text over thearrows is as follows: 1, 3′-O-TCO-SS-dATP, 3′-O-PBA-SS-dCTP,3′-O-Biotin-SS-dGTP, 3′-O-N3-SS-dTTP, DNA Polymerase. 2. Rox-Tetrazine,cy5-Streptravidin, Atexa488-SHA, R6G-DBCO, Washing, Imaging. 3. THPCleavage. 4. Repeat steps 1, 2 and 3 For Subsequent Cycles ofSequencing.

FIG. 8. Structures of Fluorescent (Cy5) Dendrimer Conjugated Tetrazine(A and B) and 3′-O-TCO-SS(DTM)-dNTPs. Incorporation of each of the four3′-O-TCO-SS(DTM)-dNTPs into the growing DNA strand in the polymerasereaction terminates the DNA synthesis, leading to the DNA products thathave a TCO group as a 3′ end. Coupling of the DNA products that have aTCO group as a 3′ end with either Molecule A or Molecule B (shown above)that has the Tetrazine moiety through the TCO-Tetrazine ligation allowsthe DNA product to be labeled with multiple fluorescent dyes, therebyfacilitating signal amplification for detection to perform either SBS atthe single-molecule level or at an ensemble level (following a schemasimilar to the one shown in FIGS. 3A-3B).

FIG. 9. Example of a Peptide-Based Fluorescent (Cy5) DendrimerConjugated Tetrazine (Molecule A) and Polymer Conjugated Tetrazine(Molecule B). Incorporation of each of the four 3′-O-TCO-SS(DTM)-dNTPsinto the growing DNA strand in the polymerase reaction terminates theDNA synthesis, leading to the DNA products that have a TCO group as a 3′end. Coupling of the DNA products that have a TCO group as a 3′ end witheither Molecule A or Molecule B (shown above) that has the Tetrazinemoiety through the TCO-Tetrazine ligation allows the DNA product to belabeled with multiple fluorescent dyes, thereby facilitating signalamplification for detection to perform either SBS at the single-moleculelevel or at an ensemble level (following a schema similar to the oneshown in FIGS. 3A-3B).

FIGS. 10A-10D. Examples of FRET Cassette Labeled Binding Molecules. FRETcassette provides numerous distinct FRET signal signatures by alteringthe distance between donor and accepter fluorophores. Binding moleculesconjugated to such FRET cassette with four unique FRET signal signaturesenables the coupling of such FRET cassette to 3′-end of the DNAextension product using “anchor” moiety coupling reaction; this allowsfor the use of two different fluorescent dyes with distinct emissionsthrough FRET to perform scarless 2-color SBS to identify the four DNAbases. In the set of FRET cassette labeled binding molecules shownabove, Rox and Cy5, serving as donor and accepter respectively, areattached with 7 or 3 dSpacer monomers to yield two different FRETcassettes: FRET Cassette A (Rox-7-Cy5 attached to SHA), which has a longseparation distance of 7 dSpacer monomers between Rox and Cy5, will havea less efficient energy transfer from the donor (Rox) to the accepter(Cy5), thereby generating a weak Cy5 emission signal and a strong Roxemission signal. FRET Cassette B (Rox-3-Cy5 attached totrans-cyclooctene TCO), which has a short separation distance of 3dSpacer monomers between Rox and Cy5, will have a more efficient energytransfer from the donor (Rox) to the accepter (Cy5), thereby generatinga strong Cy5 signal and a weak Rox signal. In Labeling Molecule C, wherethe single Rox is attached to Tetrazine, only the Rox signal isdetectable. In Labeling Molecule D, where the single Cy5 is attached toStreptavidin, only the Cy5 signal is detectable. Following a schemesimilar to the one indicated in FIG. 7 to perform SBS by carrying outthe following steps to sequence DNA: Addition of the DNA polymerase andthe four 3′-O-“anchor”-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP,3′-O-PBA-SS-dCTP, 3′-O-Biotin-SS-dGTP and 3′-O—N₃-SS-dTTP) to theimmobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. After washing away the unincorporated nucleotideanalogues, add the dye labeled binding molecules (A, B, C, D), whichwill specifically connect with each of the four unique “anchor” moietiesat the 3′-end of each DNA extension product to enable the labeling ofeach DNA product terminated with each of the four nucleotide analogues(A, C, G, T) with four distinct fluorescent signatures. Detection of theunique fluorescent signatures from the labeled DNA products allows forthe identification of the incorporated nucleotide. Next, treatment ofthe DNA products with THP cleaves the SS linker, leading to the removalof the fluorescent label and the regeneration of a free 3′-OH group onthe DNA extension product, which is ready for next cycle of DNAsequencing reaction.

FIG. 11. General Scheme of FRET Cassette Labeled Binding Molecules(e.g., SHA, Tetrazine, DBCO, Streptavidin, etc.). The FRET Cassetteprovides numerous distinct FRET signal signatures (A, B, C, D) byaltering the distance between the donor and the accepter fluorophores.Following a scheme similar to the one indicated in FIG. 7 to perform SBSby carrying out the following steps to sequence DNA: Addition of the DNApolymerase and the four 3′-O-“anchor”-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP,3′-O-PBA-SS-dCTP, 3′-O-Biotin-SS-dGTP and 3′-O—N₃-SS-dTTP) to theimmobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. After washing away the unincorporated nucleotideanalogues, add the dye labeled binding molecules (A, B, C, D), whichwill specifically connect with each of the four unique “anchor” moietiesat the 3′-end of each DNA extension product to enable the labeling ofeach DNA product terminated with each of the four nucleotide analogues(A, C, G, T) with four distinct fluorescent signatures. Detection of theunique fluorescent signatures from the labeled DNA products allows forthe identification of the incorporated nucleotide. Next, treatment ofthe DNA products with THP cleaves the SS linker, leading to the removalof the fluorescent label and the regeneration of a free 3′-OH group onthe DNA extension product, which is ready for next cycle of DNAsequencing reaction.

FIG. 12. Example Structures of 3′-O-Dye-SS(DTM)-dNTPs(3′-O-Rox-t-Butyldythiomethyl-dATP &3′-O-BodipyFL-t-Butyldythiomethyl-dCTP); 3′-O-Anchor-SS(DTM)-dNTPs(3′-O-TCO-t-Butyldythiomethyl-dGTP &3′-O-Azido-t-Butyldythiomethyl-dTTP), with their corresponding dyelabeled binding molecules (Rox Labeled Tetrazine & BodipyFL LabeledDibenzocyclooctyne).

FIGS. 13A-13B. Use of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP &3′-O-BodipyFL-SS-dCTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O—N₃-SS-dTTP &3′-O-TCO-SS-dGTP) and their corresponding dye labeled binding molecules(Rox-Tetrazine & BodipyFL-Dibenzocyclooctyne) to perform 2-color DNASBS. Addition of the DNA polymerase and the four nucleotide analogues(3′-O-Rox-SS-dATP, 3′-O-BodipyFL-SS-dCTP, 3′-O—N₃-SS-dTTP and3′-O-TCO-SS-dGTP) to the immobilized primed DNA template enables theincorporation of the complementary nucleotide analogue to the growingDNA strand to terminate DNA synthesis (STEP 1). After washing away theunincorporated nucleotide analogues, detect the fluorescent signal fromRox and BodipyFL to identify the incorporate nucleotide as A (labeledwith Rox) and C (labeled with BodipyFL). Next, add the dye labeledbinding molecules (Rox-Tetrazine & BodipyFL-Dibenzocyclooctyne) to theDNA extension products (STEP 2), which will specifically connect withthe two unique “anchor” moieties (TCO and N₃) at the 3′-end of each DNAextension product, to enable the labeling of each DNA product terminatedwith each of the two nucleotide analogues (G and T) with two distinctfluorescent dyes (labeled with Rox for G and labeled with BodipyFL forT). Detection of the unique, newly produced florescence signal from Roxand BodipyFL on the DNA extension products (in addition to the signalfrom STEP 1), allows for the identification of the newly-incorporatednucleotides as G and T respectively. Next, treatment of the DNA productswith THP cleaves the SS linker, leading to the removal of thefluorescent dye and the regeneration of a free 3′-OH group on the DNAextension product (STEP 3), which is ready for the next cycle of DNAsequencing reaction (as shown in the subsequent steps of FIGS. 13A-13B).The text in FIG. 13A is as follows: 3′-O—ROX-SS-dATP,3′-O-BodipyFl-SS-dCTP, 3′-O—N₃-SS-dGTP, 3′-O-TCO-SS-dGTP, DNAPolymerase. The text in FIG. 13B is as follows: Repeat steps 1, 2 and 3For Subsequent Cycles of Sequencing.

FIG. 14. Structures of Labeled Binding Molecules Conjugated withFluorescent Dyes via Different Cleavable t-butyldithiomethyl moiety s(which are highlighted in parentheses in this figure). Tetrazine istethered to ATTO647N via an azo linkage(Tetrazine-Azo(linker)-ATTO647N), which can be cleaved by sodiumdithionite (Na₂S₂O₄); Streptavidin is tethered to ATTO647N via adimethylketal linkage (Streptavidin-Dimethylketal(linker)-ATTO647N)),which can be cleaved under weak acidic conditions such as a citric acidbuffer (pH 4); SHA is tethered to ATTO647N via a photocleavablenitrobenzyl linkage (SHA-2-Nitrobenzyl(linker)-ATTO647N), which can becleaved by photoirradiation; DBCO is tethered to ATTO647N via an allyllinkage (Dibenzocyclooctyne-Allyl(linker)-ATTO647N), which can becleaved by Pd(0); DBCO can also be tethered to ATTO647N via Dde linkage(Dibenzocyclooctyne-Dde(linker)-ATTO647N), which can be cleaved byhydrazine. ATTO647N labeled Streptavidin (Streptavidin-ATTO647N) canalso be used in combination with three other binding moleculesconjugated with fluorescent dyes via different cleavablet-butyldithiomethyl moietys.

FIG. 15. Sample Structures of 3′-O-“anchor”-SS(DTM)-dNTPs(3′-O—N₃-SS-dATP, 3′-O-TCO-SS-dTTP, 3′-O-Biotin-SS-dCTP) along withtheir corresponding labeled binding molecules[DBCO-Azo(—N═N-Linker)-ATTO647N, Tetrazine-Dde(Linker)-ATTO647N, andStreptavidin-ATTO647N] conjugated with one florescent dye via differentcleavable linkage in combination with 3′-O-t-Butyl-SS(DTM)-dGTP(3′-O-SS-dGTP) for performing one-color SBS at the single-molecule levelor at the ensemble level.

FIGS. 16A-16C. (1) In presence of DNA polymerase, three 3′-anchornucleotides [3′-SS(DTM)N3-dATP, 3′-SS(DTM)TCO-dTTP,3′-SS(DTM)Biotin-dCTP] and 3′-tButyl-SS(DTM)-dGTP, as shown in FIG. 15]are added to the primed DNA templates to allow incorporation into theprimer; (2) Attach the fluorescent label (ATTO647N, for example) byadding DBCO-Azo-(—N═N-Linker)-ATTO647N, Tetrazine-Dde(Linker)-ATTO647N,Streptavidin-ATTO647N (as shown in FIG. 15) to the DNA extensionproducts that contain the incorporated 3′-anchor nucleotide analogues,which leads to the labeling of all the incorporated nucleotides (exceptG) at their 3′-end due to specific anchor-binding molecule interaction;(3) After washing, the first round of imaging is performed, and the DNAproducts terminated with A, C and T all display the same color, whilethe DNA products that do not emit a signal is terminated by a nucleotideG; (4) The first cleavage (I) is conducted by treatment with sodiumdithionite (Na₂S₂O₄), which only cleaves the azo linkage to remove thefluorescent dye from the DNA products terminated with the A nucleotide.The second round of imaging is performed. If the fluorescent signaldisappears after the cleavage I, the DNA products are determined ashaving incorporated an A nucleotide; (5) The second cleavage (II) isconducted by treatment with hydrazine (N₂H₄), which will cleave the Ddelinkage to remove the fluorescent dye from the DNA products terminatedwith the T nucleotide. The third round of imaging is performed. If thefluorescent signal disappears after the cleavage II, the DNA productsare determined as having incorporated a T nucleotide. The DNA productswith unchanged fluorescent signals are identified by inference as beingterminated by a C nucleotide; (6) The third cleavage (III) is conductedwith THP to cleave the disulfide bond and remove the dye on C, so thechange of the signal after the THP treatment also determines the DNAproducts as being terminated by a C nucleotide. Meanwhile, the THPtreatment will also cleave the DTM (SS) bond to regenerate free 3′-OH onall the DNA extension products, which are ready for subsequent cycles ofsingle-color DNA SBS. (7) Repeat steps 1 to 6 to continue subsequentcycles of single-color DNA SBS. The text over the arrows is as follows:FIG. 16A: 1. 3′-O—N₃-SS-dATP, 3′-O-TCO-SS-dTTP, 3′-O-Biotin-SS-dCTP,3′-O-SS-dGTP, DNA Polymerase. 2. DBCO-Azo-ATT0647N,Tetrazinc-Dde-ATT0647N, Streptavidin-ATTO0647N, Washing, Imaging. FIG.16B: 3. Cleavage 1 with Na₂S₂O₄. Washing, Imaging. 4. Cleavage II withN₂H₄ Washing, Imaging. FIG. 16C: Cleavage III with THP Washing, Imaging.

FIG. 17. Sample Structures of 3′-O-Dye-SS(DTM)-dNTP (3′-O-Rox-SS-dATP),3′-O-“anchor”-SS(DTM)-dNTPs (3′-O—N₃-SS-dTTP and 3′-O-Biotin-SS-dCTP)along with their corresponding labeled binding molecules[DBCO-Azo(—N═N-Linker)-Rox and Streptavidin-Rox] conjugated with oneflorescent dye via different cleavable linkage in combination with3′-O-t-Butyl-SS(DTM)-dGTP (3′-O-SS-dGTP) for performing one-color SBS atthe single-molecule level or at the ensemble level.

FIGS. 18A-18C. (1) In presence of DNA polymerase, two 3′-anchornucleotides [(3′-O—N₃-SS(DTM)-dTTP, 3′-O-Biotin-SS(DTM)-dCTP)],3′-O-Rox-SS(DTM)-dATP and 3′-O-tButyl-SS(DTM)-dGTP, as shown in FIG. 171are added to the primed DNA templates to allow incorporation into theprimer; (2) After washing, the first round of imaging is performed, andthe DNA products terminated with an A nucleotide analogue display theRox signal and therefore are determined as having incorporated an Anucleotide, while the other DNA products terminated at G, C, T will notdisplay any fluorescent signals; (3) Attach the fluorescent label (Rox,for example) by adding DBCO-Azo-(—N═N-Linker)-Rox, Streptavidin-Rox (asshown in FIG. 17) to the DNA extension products that contain theincorporated 3′-anchor nucleotide analogues, which leads to the labelingof all the incorporated nucleotides (except G) at their 3′-end due tospecific anchor-binding molecule interaction; (4) After washing, thesecond round of imaging is performed, and the DNA products terminatedwith A. C and T all display the same Rox signal, while the DNA productsthat do not emit a signal is terminated by a nucleotide G; (5) The firstcleavage (I) is conducted by treatment with sodium dithionite (Na₂S₂O₄),which only cleaves the azo linkage to remove the fluorescent dye Roxfrom the DNA products terminated with the T nucleotide. The second roundof imaging is performed. If the Rox fluorescent signal disappears afterthe cleavage I, the DNA products are determined as having incorporated aT nucleotide; (6) The second cleavage (II) is conducted with THP tocleave the disulfide bond and remove the dye from the DNA extensionproducts terminated with nucleotides A and C, so the change of thesignal after the THP treatment determines the DNA products as beingterminated by a C nucleotide, because DNA products as being terminatedby an A nucleotide have already being determined in the first round ofimaging described above. Meanwhile, the THP treatment will also cleavethe DTM (SS) bond to regenerate free 3′-OH on all the DNA extensionproducts, which are ready for subsequent cycles of single-color DNA SBS.Repeat steps 1 to 6 to continue subsequent cycles of single-color DNASBS. The text over the arrows is as follows: FIG. 18A: 1.3′-O-Rox-SS-dATP, 3′-O-Biotin-SS-dCTP, 3′-O—N₃-SS-dTTP, 3′-O-SS-dGTP.DNA Polymerase. Washing, Imaging. 2. Streptavidin-Rox, DBCO-Azo-Rox,Washing, Imaging. FIG. 18B: 3. Cleavage 1 with Na₂S₂O₄ Washing, Imaging.4. Cleavage II with THP Washing, Imaging. FIG. 18C: Repeat steps 1, 2,3, 4 For Subsequent cycles of Sequencing.

FIGS. 19A-19B. Sample Structures of 3′-O-Dye-SS(DTM)-dNTP(3′-O-Rox-SS-dATP), 3′-O-“anchor”-SS(DTM)-dNTPs (3′-O-TCO-SS-dTTP,3′-O-Biotin-SS-dCTP and 3′-O—N₃-SS-dGTP) along with their correspondinglabeled binding molecules [Tetrazine-Dde(Linker)-Rox, Streptavidin-Roxand DBCO-Azo(—N═N-Linker)-Rox] conjugated with one florescent dye viadifferent cleavable linkage for performing one-color SBS at thesingle-molecule level or at the ensemble level.

FIGS. 20A-20C. (1) In presence of DNA polymerase, three 3′-anchornucleotides [3′-O—N₃-SS(DTM)-dGTP, 3′-O-Biotin-SS(DTM)-dCTP,3′-O-TCO-SS(DTM)-dTTP)] and 3′-O-Rox-SS(DTM)-dATP, as shown in FIGS.19A-19B] are added to the primed DNA templates to allow incorporationinto the primer; (2) After washing, the first round of imaging isperformed, and the DNA products terminated with an A nucleotide analoguedisplay the Rox signal and therefore are determined as havingincorporated an A nucleotide, while the other DNA products terminated atG, C, T will not display any fluorescent signals; (3) Attach thefluorescent label (Rox, for example) by addingDBCO-Azo-(—N═N-Linker)-Rox, Tetrazine-Dde-Rox and Streptavidin-Rox (asshown in FIGS. 19A-19B) to the DNA extension products that contain theincorporated 3′-anchor nucleotide analogues, which leads to the labelingof all the incorporated nucleotides at their 3′-end due to specificanchor-binding molecule interaction; (4) After washing, the second roundof imaging is performed, and the DNA products terminated with A, G, T, Call display the same Rox signal. Subtraction of the Rox signals from theDNA products determined in the first round of imaging as terminated atan A nucleotide reveals the DNA products terminated at G, T, C; (5) Thefirst cleavage (1) is conducted by treatment with sodium dithionite(Na₂S₂O₄), which only cleaves the azo linkage to remove the fluorescentdye Rox from the DNA products terminated with the G nucleotide. Thesecond round of imaging is performed. If the Rox fluorescent signaldisappears after the cleavage I, the DNA products are determined ashaving incorporated a G nucleotide; (6) The second cleavage (II) isconducted with hydrazine (N₂H₄), which will cleave the Dde linkage toremove the fluorescent dye Rox from the DNA products terminated with theT nucleotide. The third round of imaging is performed. If the Roxfluorescent signal disappears after the cleavage II, the DNA productsare determined as having incorporated a T nucleotide. If the Roxfluorescent signal stays after the cleavage II, the DNA products aredetermined as having incorporated a C nucleotide; (7) The third cleavage(III) is conducted with THP to cleave the disulfide bond and remove theRox dye from the DNA extension products terminated with nucleotides Aand C, so the change of the signal after the THP treatment alsodetermines the DNA products as being terminated by a C nucleotide,because DNA products as being terminated by an A nucleotide have alreadybeing determined in the first round of imaging described above.Meanwhile, the THP treatment will also cleave the DTM (SS) bond toregenerate free 3′-OH on all the DNA extension products, which are readyfor subsequent cycles of single-color DNA SBS. Repeat steps 1 to 7 tocontinue subsequent cycles of single-color DNA SBS. The text under thearrows is as follows: FIG. 20A: 1. 3′-O-Rox-SS-dATP,3′-O-Biotin-SS-dCTP, 3′-O-TCO-dTTP, 3′-O—N₃-SS-dGTP, DNA Polymerase,Washing, Imaging. 2. Streptavidin-Rox, DBCO-Azo-Rox, Tetrazine-Dde-ROX,Washing, Imaging. FIG. 20B: 3. Cleavage 1 with Na₂S₂O₄ Washing, Imaging.4. Cleavage II with THP Washing, Imaging. FIG. 20C: 5. Cleavage III withTHP, Washing, Imaging. Repeat steps 1, 2, 3, 4, 5 For Subsequent cyclesof Sequencing.

FIG. 21. Structures of 3′-O-Linker-Label-dNTPs [3′-O-Rox-SS(DTM)-dATP,3′-O-Rox-Allyl-dTTP, 3′-O-Rox-Nitrobenzyl-dCTP] and 3′-O-SS(DTM)-dGTP.

FIG. 22. (1) In presence of DNA polymerase, the three3′-O-CleavableLinker-Label-dNTPs [3′-O-Rox-SS(DTM)-dATP,3′-O-Rox-Allyl-dTTP, 3′-O-Rox-Nitrobenzyl-dCTP] and 3′-O-tButyl-SS-dGTP,as shown in FIG. 21] are added to the primed DNA templates to allowincorporation into the primer; (2) After washing, the first round ofimaging is performed, and the DNA products terminated with C, T and Aall display the same Rox signal, while the DNA products that do not emita signal is terminated by a nucleotide G; (3) The first cleavage (1) isconducted by photo-irradiation at ˜350 nm to remove the fluorescent dyeRox from the DNA products terminated with the C nucleotide. The secondround of imaging is performed. If the Rox fluorescent signal disappearsafter the cleavage I, the DNA products are determined as havingincorporated a C nucleotide; (4) The second cleavage (II) is conductedwith Pd (0), which will cleave the allyl linkage to remove thefluorescent dye Rox from the DNA products terminated with the Tnucleotide. The third round of imaging is performed. If the Roxfluorescent signal disappears after the cleavage II, the DNA productsare determined as having incorporated a T nucleotide. If the Roxfluorescent signal stays after the cleavage II, the DNA products aredetermined as having incorporated an A nucleotide; (5) The thirdcleavage (III) is conducted with THP to cleave the disulfide bond andremove the Rox dye from the DNA extension products terminated withnucleotides A, so the change of the signal after the THP treatment alsodetermines the DNA products as being terminated by an A nucleotide.Meanwhile, the THP treatment will also cleave the DTM (SS) bond toregenerate free 3′-OH on all the DNA extension products, which are readyfor subsequent cycles of single-color DNA SBS. Repeat steps 1 to 5 tocontinue subsequent cycles of single-color DNA SBS. The text above thearrow is as follows: 1. 3′-O-SS-dGTP, 3′-O-Rox-SS-dATP,3′-O-Rox-Allyl-dTTP, 3′-O-Rox-Nitrobenzyl-dCTP, DNA Polymerase, Washing,Imaging. 2. Cleavage 1 Photo-irradiation, Washing, Imaging. 3. CleavageII, Palladium/TPPTS, Washing. Imaging. 4. Cleavage III THP Washing.Imaging. Repeat steps 1, 2, 3 and 4 For Subsequent cycles of Sequencing.

FIGS. 23A-23B. MALDI-TOF mass spectra of DNA extension products frompolymerase reactions using 3′-O-Rox-SS-dATP for 5, 10, and 30 cycles.About 50% of the primers were extended with 3′-O-Rox-SS-dATP after 5cycles. About 80% of the primers were extended after 10 cycles, andprimer was completely extended after 30 cycles.

FIG. 24. MALDI-TOF mass spectrum of DNA extension product frompolymerase reactions using 3′-O-tButyl-SS-dATP shows that extension iscompleted after 5 cycles of extension.

FIGS. 25A-25C. MALDI-TOF mass spectrum of DNA extension products frompolymerase reactions using a mixture of 3′-O-tButyl-SS-dATP and3′-O-Rox-SS-dATP at a 1:1 ratio. The extension reaction is completedafter 5 cycles and the height of the extension product peak with3′-O-tButyl-SS-dATP (Extension Product 1, M.W. 6532) is more than twicethat of the height of the extension product with 3′-O-Rox-SS-dATP(Extension Product 2. M.W. 7064), indicating that 3′-O-tButyl-SS-dATPmodified with a relatively smaller 3′-O blocking group is incorporatedby polymerase with a much higher efficiency than 3′-O-Rox-SS-dATPlabeled with a bulky Rox dye. FIG. 25B: 3′-O-tButyl-SS-dATP (M.W. 625).FIG. 25C: 3′-O-Rox-SS-dATP (M.W. 1157).

FIG. 26. MALDI-TOF mass spectrum of a DNA extension product frompolymerase reaction using 3′-O-TCO-SS-dTTP. The result shows that primeris completed extended by the 3′-O-TCO-SS-dTTP after 38 cycles to yieldan extension product at 5765 Daltons (calculated M.W. 5767).

FIGS. 27A-27B. MALDI-TOF mass spectrum of a DNA extension product frompolymerase reaction using 3′-O-Biotin-dCTP. The majority of the primer(M.W. 5136) was extended to produce a single extension product detectedat 5801 Daltons (calculated M.W. 5811).

FIGS. 28A-28C. MALDI-TOF mass spectrum of DNA extension products frompolymerase reaction using with a mixture of 3′-O-Rox-SS-dATP and3′-O-Rox-PEG₄-SS-dATP at a 1:1 ratio. The peak of the extension product2 with 3′-O-Rox-PEG₄-SS-dATP at 7311 Daltons (calculated M.W. 7314) ismuch higher than that of the extension product 1 with 3′-O-Rox-SS-dATPat 7063 Daltons (calculated M.W. 7064). This result indicates thenucleotide analogue modified by a Rox through a PEG4 linker is a bettersubstrate for the DNA polymerase than the nucleotide analogue modifiedby Rox without a PEG linker.

FIG. 29. Structures of 3′-O-t-Butyldithiomethyl-dNTPs.

FIGS. 30A-30D. Structures of four 3′-O-Dye-DTM-dNTPs. FIG. 30A:3′-O-Alexa488-t-Butyldithiomethyl-dCTP. FIG. 30B:3′-O-Cy5-t-Butyldithiomethyl-dGTP. FIG. 30C:3′-O-Rox-t-Butyldithiomethyl-dATP. FIG. 30D:3′-O-R6G-t-Butyldithiomethyl-dTTP.

FIGS. 31A-31D. Structures of four 3′-O-Dye-DTM-dNTPs with PEG4 betweendye and DTM.

FIG. 32. Cleavage of DNA extension product incorporated with a 3′-O-Dye(Label)-DTM-dNTP generates a free 3′-OH group and an extended DNA strandwithout any modification.

FIGS. 33A-33E. Experimental scheme of consecutive DNA polymeraseextension and cleavage using 3′-O-Rox-DTM-dATP as a reversibleterminator. MALDI-TOF MS spectra of the first extension (Product 1,calc. M.W. 7076), the first cleavage (Product 2, calc. M.W. 6400), andthe second extension (Product 3, calc. M.W. 7382).

FIGS. 34A-34C. DNA polymerase extension and cleavage using3′-O-Rox-PEG₄-DTM-dATP as a reversible terminator. MALDI-TOF MS spectraof the extension product and the cleavage product.

FIGS. 35A-35C. DNA polymerase extension and cleavage using3′-O-Bodipy-DTM-dTTP as a reversible terminator. MALDI-TOF MS spectra ofthe extension product and the cleavage product.

FIGS. 36A-36C. DNA polymerase extension and cleavage using3′-O-Bodipy-PEG₄-DTM-dTTP as a reversible terminator. MALDI-TOF MSspectra of the extension product and the cleavage product.

FIG. 37: Anchor and binding moieties which react covalently or formcomplexes with each other.

FIG. 38: Structures of four 3′-O-Anchor-SS(DTM)-dNTPs.

FIG. 39: Structures of three 3′-O-Anchor-SS (DTM)-dNTPs and3′-O-SS(DTM)-dATP.

FIG. 40: Example structures of four nanotags tethered to bindingmolecules, which will give distinctive current blockade signals uponattaching to the anchor moieties in NanoSBS. The nanotags can be basedon modified oligonucleotides, peptides, polyethylene glycols (PEG) or acombination thereof.

FIG. 41: Structures of nanotag conjugated binding molecules which willreact with the anchor moieties attached to the 3′-O-SS-linkernucleotides.

FIG. 42: Structures of two 3′-O-Anchor-2NB(2-NitroBenzyl)-dNTPs (top)and two 3′-O-Anchor-SS(DTM)-dNTPs (bottom) used in 2-Tag nanopore SBS.

FIG. 43: Synthetic scheme for making Tetrazine labeled TAG1.Commercially available Tetrazine NHS ester is coupled with aminomodified oligo Tag 1 yielding the Tag1-Tetrazine conjugate.

FIG. 44: Synthetic scheme for making SHA (Salicylhydroxamic acid)labeled TAG2. The amino derivative of SHA is reacted with succinicanhydride giving the acid derivative of SHA, which is converted to theNHS ester by reaction with N-hydroxysuccinimide and DCC. The SHA NHSester can then be coupled to amino modified oligo Tag2 to yield theTag2-SHA conjugate.

FIG. 45: Synthetic scheme for making Ni Bis(dithiolene) labeled TAG3.Incubating Ni Bis(dithiolene) acid with amino modified oligo Tag3 inpresence of EDC gives the Tag3-Ni Bis(dithiolene) conjugate.

FIG. 46: Synthetic scheme for making DBCO labeled TAG4. Commerciallyavailable DBCO NHS ester is coupled with amino modified oligo Tag 4yielding the Tag4-DBCO conjugate.

FIG. 47: Construction of nanopore-polymerase-DNA duplex complex (A) andnanopore-DNA duplex complex (B) for SBS on nanopore using3′-O-anchor-DTM-dNTPs and labeled binding molecules. In (B), polymeraseis added to the complex in solution (not shown).

FIG. 48A-48B: Single-molecule SBS by a nanopore using3′-O-Anchor-cleavable linker nucleotides; 4 anchor 4 tag scheme startingfrom DNA polymerase-nanopore conjugate. To the nanopore-polymerase-DNAduplex complex shown here as an example, 1) 3′-O-PBA-SS-dATP,3′-O-quadricyclane(QC)-SS-dCTP, 3′-O-TCO-SS-dGTP and 3′-O—N₃-SS-dTTP areadded, complementary 3′-O—N₃-SS-dTTP is incorporated by DNA polymerase,2) Adding the 4 tag labeled binding molecules (Tetrazine-TAG1, SHA-TAG2,Ni-bis(dithiolene)-TAG3 and DBCO-TAG4). Only Tag4 is attached to the 3′end of T due to orthogonal interaction between N₃ and DBCO, 3)Subsequent nanopore electronic detection only shows the Tag4 signal,indicating incorporation of dTTP. 4) Cleavage using TCEP or THP removesthe Tag from the 3′end, and at the same time regenerates the free 3′OHin preparation for the next cycle of sequencing. Washing steps arecarried out after each step in the procedure. Steps 1) and 2) arerepeated. Only 3′-O-TCO-SS-dGTP is incorporated and Tetrazine-TAG1 isattached, leading to 3) detection of the Tag1 signal, indicatingincorporation of G. Cleavage using TCEP or THP removes Tag1 from the3′end, again regenerating a free 3′OH. Steps 1) and 2) are repeated.3′-O-QC-SS-dCTP is incorporated and Ni-bis(dithiolene)-TAG3 is attached,leading to 3) detection of the Tag3 signal, indicating incorporation ofC. Steps 1) and 2) are repeated. 3′-O-PBA-SS-dATP is incorporated andSHA-TAG2 is attached to the 3′end, 3) nanopore electronic detectiongives a Tag2 signal, indicating incorporation of A.

FIGS. 49A-49B. Single-molecule SBS by a nanopore using3′-O-Anchor-cleavable linker nucleotides; 3 anchor 3 tag scheme startingfrom DNA polymerase-nanopore conjugate. To the nanopore-polymerase-DNAduplex complex shown here as an example, 1) 3′-O-SS-dATP,3′-O-PBA-SS-dCTP, 3′-O-TCO-SS-dGTP and 3′-O—N₃-SS-dTTP are added.Complementary 3′-O—N-SS-dTTP is incorporated by DNA polymerase, 2) the 3tag labeled binding molecules (Tetrazine-TAG1, SHA-TAG2 and DBCO-TAG3)are added. Only Tag3 is attached to the 3′ end of T due to orthogonalinteraction between N₃ and DBCO, 3) Subsequent nanopore electronicdetection only shows the Tag3 signal, indicating incorporation of dTTP.4) Cleavage using or TCEP or THP removes the Tag from the 3′end, and atthe same time regenerates the free 3′OH in preparation for the nextcycle of sequencing. Washing steps are carried out after each step inthe procedure. Steps 1) and 2) are repeated. Only 3′-O-TCO-SS-dGTP isincorporated and Tetrazine-TAG1 is attached, leading to 3) detection ofthe Tag1 signal, indicating incorporation of G. Cleavage using TCEP orTHP removes Tag1 from the 3′end, again regenerating a free 3′OH.Steps 1) and 2) are repeated. 3′-O-SS-dATP is incorporated and no tagshould be attached to the 3′end of A, therefore 3) nanopore electronicdetection shows no tag signal, indicating incorporation of A. Steps 1)and 2) are repeated. 3′-O-PBA-SS-dCTP is incorporated and SHA-TAG2 isattached, leading to 3) detection of the Tag2 signal, indicatingincorporation of C.

FIGS. 50A-50B. Single-molecule SBS by a nanopore using3′-O-Anchor-cleavable linker nucleotides; 4 anchor 4 tag scheme startingfrom DNA primer-nanopore conjugate. To the nanopore-primer complex shownhere as an example, 1) DNA polymerase, 3′-O-PBA-SS-dATP,3′-O-QC-SS-dCTP, 3′-O-TCO-SS-dGTP and 3′-O—N₃-SS-dTTP are added.Complementary 3′-O—N₃-SS-dTTP is incorporated by DNA polymerase, 2)Adding the 4 tag labeled binding molecules (Tetrazine-TAG1, SHA-TAG2,Ni-bis(dithiolene)-TAG3 and DBCO-TAG4). Only Tag4 is attached to the 3′end of T due to orthogonal interaction between N₃ and DBCO, 3)Subsequent nanopore electronic detection only shows the Tag4 signal,indicating incorporation of dTTP. 4) Cleavage using TCEP or THP removesthe Tag from the 3′end, and at the same time regenerates the free 3′OHin preparation for the next cycle of sequencing. Washing steps arecarried out after each step in the procedure. Steps 1) and 2) arerepeated. Only 3′-O-TCO-SS-dGTP is incorporated and Tetrazine-TAG1 isattached, leading to 3) detection of the Tag1 signal, indicatingincorporation of G. Cleavage using TCEP or THP removes Tag1 from the3′end, again regenerating a free 3′OH. Steps 1) and 2) are repeated.3′-O-QC-SS-dCTP is incorporated and Ni-bis(dithiolene)-TAG3 is attached,leading to 3) detection of the Tag3 signal, indicating incorporation ofC. Steps 1) and 2) are repeated. 3′-O-PBA-SS-dATP is incorporated andSHA-TAG2 is attached to the 3′end, 3) nanopore electronic detectiongives a Tag2 signal, indicating incorporation of A

FIGS. 51A-51B. Single-molecule SBS by a nanopore using3′-O-Anchor-cleavable linker nucleotides; 3 anchor 3 tag scheme startingfrom DNA primer-nanopore conjugate. To the nanopore-primer complex shownhere as an example, 1) DNA polymerase, 3′-O-SS-dATP, 3′-O-PBA-SS-dCTP,3′-O-TCO-SS-dGTP and 3′-O—N₃-SS-dTTP are added, complementary3′-O—N₃-SS-dTTP is incorporated by DNA polymerase, 2) the 3 tag labeledbinding molecules (Tetrazine-TAG1, SHA-TAG2 and DBCO-TAG3) are added.Only Tag3 is attached to the 3′ end of T due to orthogonal interactionbetween N₃ and DBCO, 3) Subsequent nanopore electronic detection onlyshows the Tag3 signal, indicating incorporation of dTTP. 4) Cleavageusing TCEP or THP removes the Tag from the 3′end, and at the same timeregenerates the free 3′OH in preparation for the next cycle ofsequencing. Washing steps are carried out after each step in theprocedure. Steps 1) and 2) are repeated. Only 3′-O-TCO-SS-dGTP isincorporated and Tetrazine-TAG1 is attached, leading to 3) detection ofthe Tag1 signal, indicating incorporation of G. Cleavage using TCEP orTHP removes Tag1 from the 3′end, again regenerating a free 3′OH.Steps 1) and 2) are repeated. 3′-O-SS-dATP is incorporated and no tagshould be attached to the 3′end of A, therefore 3) nanopore electronicdetection shows no tag signal, indicating incorporation of A. Steps 1)and 2) are repeated. 3′-O-TBA-SS-dCTP is incorporated and SHA-TAG2 isattached, leading to 3) detection of the Tag2 signal, indicatingincorporation of C.

FIGS. 52A-52C. Single-molecule SBS by a nanopore using3′-O-Anchor-cleavable linker nucleotides: 2 anchor 2 tag scheme startingfrom DNA primer-nanopore conjugate. To the nanopore-polymerase complexshown here as an example, 1) 3′-O—N₃-SS-dATP, 3′-O-TCO-SS-dCTP,3′-O-TCO-2NB-dTTP and 3′-O—N₃₋₂NB-dGTP are added, complementary3′-O—N₃-SS-dATP (top) or 3′-O—N₃₋₂NB-dGTP (bottom) are incorporated byDNA polymerase; 2) the two tag labeled binding molecules Tetrazine-TAG1and DBCO-TAG2 are added. TAG2 is attached to the 3′ end of A or G due toorthologous interaction between N₃ and DBCO. 3) Subsequent nanoporeelectronic detection shows only the Tag2 signal, indicatingincorporation of dATP or dGTP into the growing primer strand. 4)Photocleavage using 340 nm light removes the tag from the G and restoresits 3′-OH group due to its having a 2-nitrobenzyl (2NB) cleavable group.5) Signal detection indicates either a loss of the Tag2 signal,indicating that dGTP was incorporated, or a remaining Tag2 signal,indicating incorporation of dATP. 6) Cleavage of the SS group with THPrestores the 3′-OH on the A in preparation for the second cycle. Washesare carried out after each step. Steps 1)-6) are repeated for the secondcycle of sequencing. In this case, 1) incorporation of 3′-O-TCO-SS-dCTP(top) or 3′-O-TCO-2NB-dTTP (bottom) will take place. 2) the two taglabeled binding molecules Tetrazine-TAG1 and DBCO-TAG2 are added; onlyTAG2 is attached to the 3′ end of C or T due to orthologous interactionbetween TCO and tetrazine. 3) Subsequent nanopore electronic detectionshows only the Tag1 signal, indicating incorporation of dCTP or dTTPinto the growing primer strand. 4) Photocleavage using 340 nm lightremoves the tag from the T and restores its 3′-OH group due to itshaving a 2NB cleavable group. 5) Signal detection indicates either aloss of the Tag1 signal, indicating that dTTP was incorporated or aremaining Tag1 signal, indicating incorporation of dCTP. 6) Cleavage ofthe SS group with THP restores the 3′-OH on the dCTP. Steps 1)-6) arerepeated for additional cycles of sequencing.

FIG. 53: Use of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-PEG₄-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dGTP and3′-O-Biotin-SS-dCTP) for continuous SBS with MALDI-TOF MS detection ofintermediate products. Reactions were carried out in solution withmixtures of two 3′-dye modified nucleotides (3′-SS-Rox-dATP and3′-SS-BodipyFL-dTTP) and two 3′-anchor modified nucleotides(3′-SS-Biotin-dCTP and 3′-SS-TCO-dGTP). Replicate reactions consisted of20 pmol of the 51mer template shown below, 100 pmol primer orbase-extended primers (13-16mer), 150 pmol 3′-O-Dye(Anchor)-dNTPsmixture, 2 units Therminator IX DNA polymerase and 2 mM manganese in 20μl 1× Thermo Pol buffer subjected to 38 cycles of 30 sec at 65° C. and30 sec at 45° C. Reactions from multiple replicate tubes were pooled andHPLC was used to remove unused 3′-Dye(Anchor)-dNTPs and salt and obtainpure incorporation products as verified by MALDI-TOF MS. Cleavage with100 pmol tris-hydroxypropyl phosphine (THP) for 5 min at 65° C. led torecovery of the 3′ OH. The samples were treated with OligoClean &Concentrator™ kit (ZymoResearch, USA) to remove salt and cleaved groupsand sizes of products checked by MALDI-TOF MS. The 13-mer shown belowwas used in the initial reaction. In subsequent cycles, primers extendedat the 3′ end with the base from the previous cycle were used. As shownin the scheme at the left, 4 cycles of extension (a, c, e, g) andcleavage (b, d, f, g) were conducted to add A, C, G and T to the 3′ endsof these primers (complementary to the 4 bases 5′ to the underlinedprimer binding site shown in bold letters in the template). The resultsof MALDI-TOF MS analysis confirmed that the correct nucleotides wereadded and then converted to natural nucleotides containing a free 3′-OHgroup in each cycle. Addition of the nucleotide mixture to the 13-merprimer annealed to a DNA template resulted in complete incorporation of3′-SS-PEG₄-Rox-dATP into the primer as evidenced by the single observedpeak in the mass spectrum (MS) of 5188 Da (5188 Da expected) (a). Aftertreatment with THP to cleave the 3′-SS-PEG4-Rox group, a single MS peakwas observed at 4264 Da (4272 Da expected) (b). Extension of the 14-merprimer in the second cycle revealed incorporation of 3′-SS-Biotin-dCTPinto the growing primer strand (single MS peak at 4941 Da observed, 4939Da expected) (c). After treatment with THP, a single cleavage peak at4564 Da was found (4561 Da expected) (d). In the third cycle,incorporation of 3′-SS-TCO-dGTP generated a MS peak of 5184 Da (5194 Daexpected) (e) and complete cleavage of the anchor and restoration of the3′-OH group (MS peak at 4894 Da (4890 Da expected) was shown by MS (f).Finally, in the fourth cycle, the newly formed 16-mer DNA strand wasused as a primer for 3′-SS-BodipyFL-dTTP incorporation. The MS results(g and h) demonstrated a single peak with molecular weight of 5621 Da(5620 Da expected) for 3′-SS-BodipyFL-dTTP incorporation and 5197 Da(5195 Da expected) after cleavage.

51mer template: 5′-TACATCAACTACCCGGAGGCCAAGTACGGCGGGTACGT CCTTGACAATGTG-3′ 13mer primer: 5′-CACATTGICAAGG-3′ MW: 3959After each incorporation, the expected size of the product should be thesum of the starting primer plus the incoming nucleotide minus the MW(175) of the pyrophosphate group, yielding MWs of 5188 Da, 4939 Da, 5194Da and 5620 Da.

FIG. 54: Four base read obtained using four-color approach. Using thelooped priming template shown at the top of the figure, in which thenext four bases to be added are C, A, T, C, reactions were carried outas in the protocol for FIG. 70. 5′-NH₂-modified template was immobilizedon NHS ester-modified slides from Surmodics (as described previously inthe patent). Each cycle was carried out as follows: (1) extension with60 μl of 0.02 μM 3′-O-Rox-SS-dATP, 0.05 μM 3′-O-BodipyFL-SS-dTTP, 0.5 μM3′-O-Biotin-SS-dCTP, 0.5 μM 3′-O-TCO-SS-dGTP, 1× Thermo Pol ReactionBuffer (NEB), 2 mM MnCl₂, 2-10 U Therminator IX DNA polymerase for 15min at 65° C.; (2) washing with 1× Thermo Pol Reaction Buffer; (3) chasewith 60 μl of 4 μM each of the four 3′-O-SS(DTM)-dNTPs, 1× Thermo PolReaction Buffer, 2 mM MnCl₂, 2-10 U Therminator IX DNA polymerase for 10min at 65° C.; (4) washing with 1× Thermo Pol Reaction Buffer; (5)labeling with 60 μl of 10 μM Tetrazine-PEG4-TAMRA (used as analternative to Tetraine-Cy3 in this specific experiment), 4 μMStreptavidin-Cy5, 1×PBS, pH 7.4 for 15 min at 37° C.; (6) washing with1× Thermo Pol Reaction Buffer, 1×SPSC buffer and water; (7) scanning airdried slides at 488 nm, 543 nm, 594 nm and 633 nm emission settings torecord fluorescence intensity of spots; (8) cleavage with 10 mM THP for10 min at 65° C.; (9) washing with water, 1×SPSC, and water again; (9)scanning air dried slides to determine background (repeating washes asnecessary to minimize the background). The above was carried out 4 timesto obtain the raw image intensity readings shown in the bar graph at thebottom for the first four bases of the extended primer.

FIG. 55: Structures of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-PEG4-SS-dGTPand 3′-O-Biotin-SS-dCTP) with their corresponding Dye Labeled BindingMolecules (TAMRA Labeled Tetrazine and Cy5 Labeled Streptavidin) toperform 4-color DNA SBS using approach delineated in FIG. 70.

FIG. 56: Four and six base reads obtained using two-color approach.Using the looped priming template shown at the top of the figure, inwhich the next four bases to be added are T, A, G, A, or the loopedpriming template shown in the middle of the figure, in which the nextsix bases are C, A, T, C, A, A, reactions were carried out as in theprotocol for FIG. 71. 5′-NH-12-modified template was immobilized on NHSester-modified slides from Surmodics (as described previously in thepatent). Each cycle was carried out as follows: (1) extension with 60 μlof 0.02 μM 3′-O-Rox-PEG4-SS-dATP, 0.05 μM 3′-O-BodipyFL-SS-dTTP, 0.5 μM3′-O-Biotin-SS-dCTP, 0.2 μM 3′-O-TCO-SS-dGTP, 1× Thermo Pol ReactionBuffer (NEB), 2 mM MnCl₂, 2-10 U Therminator IX DNA polymerase for 15min at 65° C.; (2) washing with 1× Thermo Pol Reaction Buffer; (3) chasewith 60 μl of 4 μM each of the four 3′-O-SS(DTM)-dNTPs, 1× Thermo PolReaction Buffer, 2 mM MnCl₂, 2-10 U Therminator IX DNA polymerase for 10min at 65° C.; (4) washing with 1× Thermo Pol Reaction Buffer; (5)scanning air dried slides at 488 nm and 594 nm emission settings torecord fluorescence intensity of spots; (6) labeling with 60 μl of 10 μMTetrazine-PEG4-Alexa488, 4 μM Streptavidin-Alexa594, 1×PBS, pH 7.4 for10 min at 37° C.; (7) washing with 1× Thermo Pol Reaction Buffer, 1×SPSCbuffer and water; (8) scanning air dried slides at 488 nm and 594 nmemission settings to record fluorescence intensity of spots; (9)cleavage with 10 mM THP for 10 min at 65° C.; (10) washing with water,1×SPSC, and water again; (11) scanning air dried slides to determinebackground (repeating washes as necessary to obtain minimal background).The above was carried out 4-6 times to obtain the raw image intensityreadings shown in the bar graphs below the template structures. In eachcycle, E represents the imaging results after the extension and Lrepresents the imaging results after the labeling. So in the top graph,the T is determined after the initial extension due to the presence ofthe BodipyFL dye directly attached to the 3′-O- of the dTTP, as are theA's in the second and fourth cycle; however the G in the third cycle isnot seen until the labeling reaction in which the Alexa488-tetrazine isconjugated to the anchoring molecule (TCO) on the 3′-O- of the dGTP.Similarly in the lower bar graph, the A's and T's are visualizedimmediately after extension, but the C's are not observed until thelabeling reaction is performed.

FIG. 57: Structures of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-PEG4-SS-dGTPand 3′-O-Biotin-SS-dCTP) with their corresponding Dye Labeled BindingMolecules (Alexa488 Labeled Tetrazine and Alexa594 Labeled Streptavidin)to perform 2-color DNA SBS using approach delineated in FIG. 71.

FIG. 58: Structures of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP) and 3′-O-SS(DTM)-dNTP-SS-Dyes(3′-O-SS-dGTP-7-SS-Cy5 and 3′-O-SS-dCTP-5-SS-R6G) for 4-color sequencingusing approach delineated in FIG. 72.

FIG. 59: Structures of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP and3′-O-Biotin-SS-dTTP), 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5and 3′-O-SS-dCTP-5-SS-R6G) and the corresponding Dye Labeled BindingMolecules (Rox Labeled Tetrazine and BodipyFL Labeled Streptavidin) toperform 4-color DNA SBS using approach delineated in FIG. 73.

FIG. 60: Structures of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP and3′-O-Biotin-SS-dTTP), 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5and 3′-O-SS-dCTP-5-SS-R6G) and the corresponding Dye Labeled BindingMolecules (Cy5 Labeled Tetrazine and R6G Labeled Streptavidin) toperform 2-color DNA SBS using approach delineated in FIG. 74.

FIG. 61: Structures of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP) and 3′-O-DTM(SS)-dNTP-Azo-Dyes(3′-O-SS-dGTP-7-Azo-Rox or 3′-O-SS-dGTP-7-SS-Azo-Rox and3′-O-SS-dCTP-5-Azo-BodipyFL or 3′-O-SS-dCTP-5-SS-Azo-BodipyFL) for2-color DNA SBS using approach delineated in FIG. 75.

FIG. 62: 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dATP-7-SS-Rox and3′-O-SS-dUTP-5-SS-BodipyFL) and 3′-O-SS(DTM)-dNTP-SS-Azo-Dyes(3′-O-SS-dGTP-7-Azo-Rox or 3′-O-SS-dGTP-7-SS-Azo-Rox and3′-O-SS-dCTP-5-Azo-BodipyFL or 3′-O-SS-dCTP-5-SS-Azo-BodipyFL) for2-color DNA SBS.

FIG. 63: 3′-O-Anchor-SS(DTM)-dNTP (3′-O-TCO-SS-dCTP and3′-O-N3-SS-dATP), 3′-O-Anchor-2NB-dNTPs (3′-O-TCO-2NB-dTTP and3′-O-N3-2NB-dGTP) and their corresponding Dye-labeled binding molecules(Rox labeled tetrazine and BodipyFl labeled DBCO) for 2-color DNA SBSusing approach delineated in FIG. 76.

FIG. 64: Structures of 3′-O-Anchor-SS(DTM)-dNTP,3′-O-Anchor-Allyl-dNTPs, and 3′-O-Anchor-2NB-dNTPs. Combinatorial use oftwo from one category with the same anchor, two from another categorywith another anchor and their corresponding two Dye-labeled bindingmolecules results in 2-color DNA SBS. One specific approach is shown inFIG. 71 as an example.

FIG. 65: Synthesis of Azo Linker and general method to synthesize3′-O-SS(DTM)-dNTP-SS-Azo-Dye. The amino acid derivative of the Azolinker molecule is synthesized using the well-established diazoniumcoupling reaction. The resulting compound is coupled with Dye NHS estergiving the dye labeled acid derivative of the Azo linker, which can befurther converted to the NHS ester by treatment with DSC and TEA. Theproduct is then coupled to the amino group of 3′-O-SS(DTM)-dNTP-SS-NH₂yielding 3′-O-SS(DTM)-dNTP-SS-Azo-Dye.

FIG. 66: Example synthesis 3′-O-SS(DTM)-dGTP-SS-Azo-Rox and3′-O-SS(DTM)-dTTP-SS-Azo-BodipyFL. Rox and BodipyFL labeled Azo LinkerNHS esters are coupled with 3′-O-SS(DTM)-dGTP-SS-NH₂ and3′-O-SS(DTM)-dTTP-SS-NH₂ giving 3′-O-SS(DTM)-dGTP-SS-Azo-Rox and3′-O-SS(DTM)-dTTP-SS-Azo-BodipyFL.

FIG. 67: Synthesis of 3′-O-SS(DTM)-dATP-SS-Rox.

FIG. 68: Synthesis of 3′-O-SS(DTM)-dUTP-SS-BodipyFL.

FIG. 69: Example syntheses of 3′-O-Anchor-2NB-dNTP(3′-O-TCO-2-Nitrobenzyl-dTTP and 3′-O-Azido-2-Nitrobenzyl-dGTP).

FIG. 70. Use of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-PEG4-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dGTP and3′-O-Biotin-SS-dCTP) with their corresponding Dye Labeled BindingMolecules (TAMRA Labeled Tetrazine and Cy5 Labeled Streptavidin) toperform 4-color DNA SBS. Step 1, addition of DNA polymerase and the fournucleotide analogues (3′-O-Rox-PEG₄-SS-dATP, 3′-O-BodipyFL-SS-dTTP,3′-O-TCO-SS-dGTP and 3′-O-Biotin-SS-dCTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand to terminate DNA synthesis. Step 2,Chase: addition of the DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS(DTM)-nucleotide analogue to the growing DNAstrands that were not extended with one of the dye or anchor labeleddNTPs in step 1. The growing DNA strands are terminated with one of thefour dye or anchor labeled nucleotide analogues (A, C, G, T) or the sameone of the four nucleotide analogues (A, C, G, T) without dye or anchor.Step 3, Next, the dye labeled binding molecules (TAMRA labeled tetrazineand Cy5 labeled streptavidin) are added to the DNA extension products,which will specifically connect with the two unique “anchor” moieties(TCO and biotin) on each DNA extension product, to enable the labelingof each DNA product terminated with each of the two nucleotide analogues(G and C) with two distinct fluorescent dyes (labeled with TAMRA for Gand labeled with Cy5 for C). Step 4, after washing away the unbound dyelabeled binding molecules, detection of the unique fluorescence signalfrom each of the fluorescent dyes on the DNA products allows theidentification of the incorporated nucleotide for sequencedetermination. Next, in Step 5, treatment of the DNA products with THPcleaves the SS linker, leading to the removal of the fluorescent dye andthe regeneration of a free 3′-OH group on the DNA extension product,which is ready for the next cycle of the DNA sequencing reaction.Structures of modified nucleotides used in this scheme are shown in FIG.55

FIG. 71. Use of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-PEG4-SS-dGTPand 3′-O-Biotin-PEG4-SS-dCTP) with their corresponding Dye LabeledBinding Molecules (Alexa488-PEG4 Labeled Tetrazine and Alexa594 LabeledStreptavidin) to perform 2-color DNA SBS. Demonstration of Successful2-Color Continuous Sequencing Using a Combination of3′-O-Dye-SS(DTM)-dNTPs and 3′-O-Anchor-SS(DTM)-dNTPs with theirCorresponding Dye Labeled Binding Molecules on Immobilized DNA Templates(Scheme Z2 and FIG. 56). Use of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATPand 3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs(3′-O-TCO-PEG4-SS-dGTP and 3′-O-Biotin-PEG4-SS-dCTP) with theircorresponding Dye Labeled Binding Molecules (Alexa488-PEG4 LabeledTetrazine and Alexa594 Labeled Streptavidin) to perform 2-color DNA SBS.Although 4 different dyes have been used in this experiment, Rox andAlexa594 have very similar absorption and emission spectra, as doBodipyFL and Alexa488. Hence this is described as a 2-color experiment.Step 1, addition of DNA polymerase and the four nucleotide analogues(3′-O-Rox-SS-dATP, 3′-O-BodipyFL-SS-dTTP, 3′-O-TCO-PEG4-SS-dGTP and3′-O-Biotin-PEG4-SS-dCTP) to the immobilized primed DNA template enablesthe incorporation of the complementary nucleotide analogue to thegrowing DNA strand to terminate DNA synthesis. Step 2, Chase: additionof the DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the growing DNA strandsthat were not extended with one of the dye or anchor labeled dNTPs instep 1. The growing DNA strands are terminated with one of the four dyeor anchor labeled nucleotide analogues (A, C, G, T) or the same one ofthe four nucleotide analogues (A, C, G, T) without dye or anchor. Step3, Next, the dye labeled binding molecules (Alexa488-PEG labeledtetrazine and Alexa594 labeled streptavidin) are added to the DNAextension products, which will specifically connect with the two unique“anchor” moieties (TCO and biotin) on each DNA extension product, toenable the labeling of each DNA product terminated with each of the twonucleotide analogues (G and C) with two distinct fluorescent dyes(labeled with Alexa488 for G and labeled with Alexa594 for C). Step 4,after washing away the unbound dye labeled binding molecules, detectionof the unique fluorescence signal from each of the fluorescent dyes onthe DNA products allows the identification of the incorporatednucleotide for sequence determination. Next, in Step 5, treatment of theDNA products with THP cleaves the SS linker, leading to the removal ofthe fluorescent dye and the regeneration of a free 3′-OH group on theDNA extension product, which is ready for the next cycle of the DNAsequencing reaction. Structures of modified nucleotides used in thisscheme are shown in FIG. 57.

FIG. 72. Use of 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5,3′-O-SS-dCTP-5-SS-R6G); 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-PEG4-SS-dATPand 3′-O-BodipyFL-SS-dTTP) for 4-color DNA SBS. Use of3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5,3′-O-SS-dCTP-5-SS-R6G); 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-PEG4-SS-dATPand 3′-O-BodipyFL-SS-dTTP) for 4-color DNA SBS. Step 1, Addition of DNApolymerase and the four nucleotide analogues (3′-O-SS-dGTP-7-SS-Cy5,3′-O-SS-dCTP-5-SS-R6G, 3′-O-Rox-PEG₄-SS-dATP and 3′-O-BodipyFL-SS-dTTP)to the immobilized primed DNA template enables the incorporation of thecomplementary dye labeled nucleotide analogue to the growing DNA strand.The growing DNA strand is terminated with each of the four nucleotideanalogues (A, C, G, T) with the four distinct fluorescent dyes. Step 2,Chase: addition of DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the subset of growing DNAstrands in the ensemble that were not extended with any of the dyelabeled dNTPs in step 1. The growing DNA strands are terminated with oneof the four nucleotide analogues (A, C, G, T) with the four distinctfluorescent dyes or the same one of the four nucleotide analogues (A, C,G, T) without dye. Step 3, after washing away the unincorporatednucleotide analogues, detection of the unique fluorescence signal fromeach of the fluorescent dyes on the DNA products allows theidentification of the incorporated nucleotide for sequencedetermination. Next, in Step 4, treatment of the DNA products with THPcleaves the SS linker, leading to the removal of the fluorescent dye andthe regeneration of a free 3′-OH group on the DNA extension product,which is ready for the next cycle of the DNA sequencing reaction.Structures of modified nucleotides used in this scheme are shown in FIG.58.

FIG. 73. Use of 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5,3′-O-SS-dCTP-5-SS-R6G); 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP,3′-O-Biotin-SS-dTTP) with their corresponding Dye Labeled BindingMolecules (Rox Labeled Tetrazine and BodipyFL Labeled Streptavidin) toperform 4-color DNA SBS. Use of 3′-O-SS(DTM)-dNTP-SS-Dyes(3′-O-SS-dGTP-7-SS-Cy5, 3′-O-SS-dCTP-5-SS-R6G);3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP, 3′-O-Biotin-SS-dTTP) withtheir corresponding Dye Labeled Binding Molecules (Rox Labeled Tetrazineand BodipyFL Labeled Streptavidin) to perform 4-color DNA SBS. Step 1,addition of DNA polymerase and the four nucleotide analogues(3′-O-SS-dGTP-7-SS-Cy5, 3′-O-SS-dCTP-5-SS-R6G, 3′-O-TCO-SS-dATP and3′-O-Biotin-SS-dTTP) to the immobilized primed DNA template enables theincorporation of the complementary nucleotide analogue to the growingDNA strand to terminate DNA synthesis. Step 2, Chase: addition of DNApolymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS(DTM)-nucleotide analogue to the subset of growingDNA strands in the ensemble that were not extended with any of the dyelabeled dNTPs in step 1. The growing DNA strands are terminated with oneof the four dye or anchor labeled nucleotide analogues (A, C, G, T) orthe same one of the four nucleotide analogues (A, C, G, T) without dyeor anchor. Step 3, next, the dye labeled binding molecules (Rox labeledtetrazine and BodipyFL labeled streptavidin) are added to the DNAextension products, which will specifically connect with the two unique“anchor” moieties (TCO and biotin) on each DNA extension product, toenable the labeling of each DNA product terminated with each of the twonucleotide analogues (A and T) with two distinct fluorescent dyes(labeled with Rox for A and labeled with BodipyFL for T). Step 4, afterwashing away the unbound dye-labeled binding molecules, detection of theunique fluorescence signal from each of the fluorescent dyes on the DNAproducts allows identification of the incorporated nucleotides forsequence determination. A Rox signal indicates incorporation of A, aBodipyFL signal indicates incorporation of T, a Cy5 signal indicatesincorporation of G and an R6G signal indicates incorporation of C. Next,in Step 5, treatment of the DNA products with THP cleaves the SS linker,leading to the removal of the remaining fluorescent dye and theregeneration of a free 3′-OH group on the DNA extension product, whichis ready for the next cycle of the DNA sequencing reaction. Structuresof modified nucleotides used in this scheme are shown in FIG. 59.

FIG. 74. Use of 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5,3′-O-SS-dCTP-5-SS-R6G); 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP,3′-O-Biotin-SS-dTTP) with their corresponding Dye Labeled BindingMolecules (Cy5 Labeled Tetrazine and R6G Labeled Streptavidin) toperform 2-color DNA SBS. Use of 3′-O-SS(DTM)-dNTP-SS-Dyes(3′-O-SS-dGTP-7-SS-Cy5, 3′-O-SS-dCTP-5-SS-R6G);3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP, 3′-O-Biotin-SS-dTTP) withtheir corresponding Dye Labeled Binding Molecules (Cy5 Labeled Tetrazineand R6G Labeled Streptavidin) to perform 2-color DNA SBS. Step 1,addition of DNA polymerase and the four nucleotide analogues(3′-O-SS-dGTP-7-SS-Cy5, 3′-O-SS-dCTP-5-SS-R6G, 3′-O-TCO-SS-dATP and3′-O-Biotin-SS-dTTP) to the immobilized primed DNA template enables theincorporation of the complementary nucleotide analogue to the growingDNA strand to terminate DNA synthesis. Step 2, Chase: addition of DNApolymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the subset of growing DNAstrands in the ensemble that were not extended with any of the dyelabeled dNTPs in step 1. The growing DNA strands are terminated with oneof the four dye labeled nucleotide analogues (A, C, G, T) or the sameone of the four nucleotide analogues (A, C, G, T) without dye. Step 3,after washing away the unincorporated dye labeled nucleotides, detectionof the unique fluorescence signal from each of the fluorescent dyes onthe DNA products allows identification of the incorporated nucleotidefor sequence determination, Cy5 signal indicates incorporation of G, R6Gsignal indicates incorporation of C. Step 4, next, the dye labeledbinding molecules (Cy5 labeled tetrazine and R6G labeled streptavidin)are added to the DNA extension products, which will specifically connectwith the two unique “anchor” moieties (TCO and biotin) on each DNAextension product, to enable the labeling of each DNA product terminatedwith each of the two nucleotide analogues (A and T) with two distinctfluorescent dyes (labeled with Cy5 for A and labeled with R6G for T).Step 5, after washing away the unattached labels, a second round ofdetection of the unique fluorescence signal from each of the fluorescentdyes on the DNA products allows the identification of the incorporatednucleotide for sequence determination. Appearance of a Cy5 signalindicates incorporation of A, R6G signal indicates incorporation of T.Next, in Step 6, treatment of the DNA products with THP cleaves the SSlinker, leading to the removal of the remaining fluorescent dye and theregeneration of a free 3′-OH group on the DNA extension product, whichis ready for the next cycle of the DNA sequencing reaction. Structuresof modified nucleotides used in this scheme are shown in FIG. 60.

FIG. 75. Use of 3′-O-SS(DTM)-dNTP-Azo-Dyes (3′-O-SS-dGTP-7-Azo-Rox,3′-O-SS-dCTP-5-Azo-BodipyFL); 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP,3′-O-BodipyFL-SS-dTTP) to perform 2-color DNA SBS. Use of3′-O-SS(DTM)-dNTP-Azo-Dyes (3′-O-SS-dGTP-7-Azo-Rox,3′-O-SS-dCTP-5-Azo-BodipyFL); 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP,3′-O-BodipyFL-SS-dTTP) to perform 2-color DNA SBS. Step 1, addition ofDNA polymerase and the four nucleotide analogues(3′-O-SS-dGTP-7-Azo-Rox, 3′-O-SS-dCTP-5-Azo-BodipyFL, 3′-O-Rox-SS-dATPand 3′-O-BodipyFL-SS-dTTP) to the immobilized primed DNA templateenables the incorporation of the complementary nucleotide analogue tothe growing DNA strand to terminate DNA synthesis. Step 2, Chase:addition of DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the subset of growing DNAstrands in the ensemble that were not extended with any of the dyelabeled dNTPs in step 1. The growing DNA strands are terminated with oneof the four dye labeled nucleotide analogues (A, C, G, T) or the sameone of the four nucleotide analogues (A, C, G, T) without dye. Step 3,after washing away the unincorporated dye labeled nucleotides, detectionof the unique fluorescence signal from each of the fluorescent dyes onthe DNA products allows the identification of the incorporatednucleotide for sequence determination. Rox signal indicatesincorporation of A or G, BodipyFL signal indicates incorporation of C orT. Step 4, cleavage of Azo linker by adding sodium dithionite (Na₂S₂O₄)to the elongated DNA strands results in removal of Rox from incorporatedG and BodipyFL from incorporated C. Step 5, after washing away thecleaved dyes, a second round of detection of the unique fluorescencesignal from each of the fluorescent dyes on the DNA products allows theidentification of the incorporated nucleotide for sequencedetermination. Disappearance of Rox signal indicates incorporation of G,and disappearance of BodipyFL signal indicates incorporation of C.Remaining Rox signal indicates incorporation of A, and remainingBodipyFL signal indicates incorporation of T. Next, in Step 6, treatmentof the DNA products with THP cleaves the SS linker, leading to theremoval of the remaining fluorescent dye and the regeneration of a free3′-OH group on the DNA extension product, which is ready for the nextcycle of the DNA sequencing reaction. The presence of an additional SSlinkage between the Azo group and the base results in the production ofa shorter scar on the incorporated nucleotide after THP treatment whichshould result in longer reads. Structures of modified nucleotides usedin this scheme are shown in FIG. 61.

FIG. 76. Use of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O—N₃-SS-dATP and3′-O-TCO-SS-dCTP) and 3′-O-Anchor-2-Nitrobenzyl-dNTPs(3′-O—N₃-2-Nitrobenzyl-dGTP and 3′-O-TCO-2-Nitrobenzyl-dTTP) with theircorresponding Dye Labeled Binding Molecules (BodipyFL Labeled DBCO andRox labeled Tetrazine) to perform 2-color DNA SBS. Use of3′-O-Anchor-SS(DTM)-dNTPs (3′-O—N₃-SS-dATP and 3′-O-TCO-SS-dCTP) and3′-O-Anchor-2-Nitrobenzyl-dNTPs (3′-O—N₃-2-Nitrobenzyl-dGTP and3′-O-TCO-2-Nitrobenzyl-dTTP) with their corresponding Dye LabeledBinding Molecules (BodipyFL Labeled DBCO and Rox labeled Tetrazine) toperform 2-color DNA SBS. Step 1, addition of DNA polymerase and the fournucleotide analogues (3′-O—N₃-SS-dATP, 3′-O-TCO-SS-dCTP,3′-O—N₃-2-Nitrobenzyl-dGTP and 3′-O-TCO-2-Nitrobenzyl-dTTP) to theimmobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. Step 2, Chase: addition of DNA polymerase and four3′-O-SS(DTM)-dNTPs (3′-O-t-Butyldithiomethyl(SS)-dATP,3′-O-t-Butyldithiomethyl(SS)-dCTP, 3′-O-t-Butyldithiomethyl(SS)-dTTP and3′-O-t-Butyldithiomethyl(SS)-dGTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary3′-O-SS(DTM)-nucleotide analogue to the subset of growing DNA strands inthe ensemble that were not extended with any of the dye labeled dNTPs instep 1. The growing DNA strands are terminated with one of the fouranchor labeled nucleotide analogues (A, C, G, T) or the same one of thefour nucleotide analogues (A, C, G, T) without dye or anchor. Step 3,next, the dye labeled binding molecules (Rox labeled Tetrazine andBodipyFL labeled DBCO) are added to the DNA extension products, whichwill specifically connect with the two unique “anchor” moieties (TCO andN₃) on each DNA extension product, to enable the labeling of each DNAproduct terminated with each of the four nucleotide analogues with oneof the two dyes (A and G with BodipyFL and C and T with Rox). Step 4,after washing away the unbound dye-labeled binding molecules, detectionof the fluorescence signals from each of the fluorescent dyes on the DNAproducts allows partial identification of the incorporated nucleotidesfor sequence determination. A BodipyFL signal indicates incorporation ofA or G, a Rox signal indicates incorporation of T or C. Next, in Step 5,treatment of the DNA products with 340 nm light cleaves the2-Nitrobenzyl linker, leading to the removal of the fluorescent dye andthe regeneration of a free 3′-OH group on the DNA extension productsextended with either a G or T. After washing, in Step 6 imaging iscarried out a second time to detect remaining fluorescent signals. Lossof a BodipyFL signal indicates that the incorporated nucleotide was a G,a remaining Bodipy FL signal indicates that the incorporated nucleotidewas an A; similarly loss of a Rox signal indicates that the incorporatednucleotide was a T, a remaining Rox signal indicates that theincorporated nucleotide was a C. Finally, in Step 7, treatment with THPcleaves any dye remaining on incorporated A or C, and restores the 3′-OHon those nucleotides as well. At this point, the extension products areready for the next cycle of the DNA sequencing reaction. Structures ofmodified nucleotides used in this scheme are shown in FIG. 63.

FIG. 77: (Scheme A) Use of 3′-O-SS(DTM)-dNTP-SS-Dyes(3′-O-SS-dGTP-7-SS-Rox, 3′-O-SS-dCTP-5-SS-Alexa488);3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP, 3′-O-Biotin-SS-dTTP) andappropriate dye labeled anchor binding molecules (Tetrazine-Rox,Streptavidin-Alexa488) to perform 2-color DNA SBS. Step 1, addition ofDNA polymerase and the four nucleotide analogues (3′-O-SS-dGTP-7-SS-Rox,3′-O-SS-dCTP-5-SS-Alexa488, 3′-O-TCO-SS-dATP and 3′-O-Biotin-SS-dTTP) tothe immobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. Step 2, Chase: addition of DNA polymerase and four3′-O-SS(DTM)-dNTPs (3′-O-t-Butyldithiomethyl(SS)-dATP,3′-O-t-Butyldithiomethyl(SS)-dCTP, 3′-O-t-Butyldithiomethyl(SS)-dTTP and3′-O-t-Butyldithiomethyl(SS)-dGTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary3′-O-SS-nucleotide analogue to the subset of growing DNA strands in theensemble that were not extended with any of the dye or anchor labeleddNTPs in step 1. The growing DNA strands are terminated with one of thefour dye or anchor labeled nucleotide analogues (A, C, G, T) or theequivalent nucleotide analogues (A, C, G, T) without dye. Step 3, afterwashing away the unincorporated dye labeled nucleotide analogues,detection of the unique fluorescence signal from each of the fluorescentdyes on the DNA products allows the precise identification of two of theincorporated nucleotide analogues for sequence determination. Rox signalindicates incorporation of G, Alexa488 signal indicates incorporation ofC. Step 4, addition of Tetrazine-Rox and Streptavidin-Alexa488 leads tolabeling of the two nucleotide analogues with 3′ anchors. Step 5, afterwashing away the excess labeling molecules, a second round of detectionof the unique fluorescence signal from each of the fluorescent dyes onthe DNA products allows the identification of the incorporatednucleotide for sequence determination. Appearance of a previouslyundetected Rox signal indicates incorporation of A, and appearance of apreviously undetected Alexa488 signal indicates incorporation of T.Next, in Step 6, treatment of the DNA products with THP cleaves the SSlinker, leading to the removal of the remaining fluorescent dye and theregeneration of a free 3′-OH group on the DNA extension product. Step 7,after washing away the THP, an optional imaging step allows confirmationof absence of any remaining fluorescent label indicating readiness forthe next cycle of the DNA sequencing reaction. Although Scheme A ispresented here as an ensemble SBS approach, it can also be used forsingle molecule SBS sequencing with an appropriate imaging setup.Structures of modified nucleotide analogues used in this scheme areshown in FIG. 78.

FIG. 78: Structures of 3′-O-DTM(SS)-dNTP-SS-Dye (3′-O-SS-dGTP-7-SS-Rox,3′-O-SS-dCTP-5-SS-Alexa488), 3′-O-Anchor-SS-dNTP (3′-O-TCO-SS-dATP,3′-O-Biotin-SS-dTTP) and the labeled binding molecules (Rox LabeledTetrazine and Alexa 488 labeled Straptavidin) for 2-color DNA SBS as inScheme A.

FIG. 79: (Scheme B) Use of 3′-O-SS(DTM)-dNTP-SS-Dye(3′-O-SS-dTTP-5-SS-BodipyFL), 3′-O-SS(DTM)-dNTP-Azo-Dyes(3′-O-SS-dCTP-5-Azo-BodipyFL, 3′-O-SS-dGTP-7-Azo-Rox), and3′-O-Dye-SS(DTM)-dNTP (3′-O-Rox-SS-dATP) to perform 2-color DNA SBS.Step 1, addition of DNA polymerase and the four nucleotide analogues(3′-O-SS-dTTP-5-SS-BodipyFL, 3′-O-SS-dCTP-5-Azo-BodipyFL,3′-O-SS-dGTP-7-Azo-Rox and 3′-O-Rox-SS-dATP) to the immobilized primedDNA template enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand to terminate DNA synthesis. Step 2,Chase: addition of DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O—I-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the subset of growing DNAstrands in the ensemble that were not extended with any of the dyelabeled dNTPs in step 1. The growing DNA strands are terminated with oneof the four dye labeled nucleotide analogues (A, C, G, T) or theequivalent nucleotide analogues (A, C, G, T) without dye. Step 3, afterwashing away the unincorporated dye labeled nucleotide analogues,detection of the fluorescence signal from the fluorescent dyes on theDNA products allows the identification of two of the incorporatednucleotide analogues for sequence determination. Rox signal indicatesincorporation of A or G, BodipyFL signal indicates incorporation ofeither C or T. Step 4, treatment with sodium dithionite cleaves the Azolinker. Step 5, after washing away the cleaved dyes, a second round ofdetection of any remaining fluorescence signal allows the identificationof the incorporated nucleotide for sequence determination. Loss of aBodipyFL signal indicates incorporation of C, remaining BodipyFL signalindicates incorporation of T. Loss of Rox signal indicates incorporationof G, remaining Rox signal indicates incorporation of A. Next, in Step6, treatment of the DNA products with THP cleaves the SS linker, leadingto the removal of the remaining fluorescent dye and the regeneration ofa free 3′-OH group on the DNA extension product. Although Scheme B ispresented here as an ensemble SBS approach, it can also be used forsingle molecule SBS sequencing with an appropriate imaging setup.Structures of modified nucleotide analogues used in this scheme areshown in FIG. 80.

FIG. 80: Structures of 3′-O-Dye-DTM(SS)-dNTPs (3′-O-Rox-SS-dATP),3′-O-DTM(SS)-dNTP-SS-Dye (3′-O-SS-dTTP-5-SS-BodipyFL) and3′-O-DTM(SS)-dNTP-SS-Azo-Dyes (3′-O-SS-dGTP-7-Azo-Rox and3′-O-SS-dCTP-5-Azo-BodipyFL) for 2-color DNA SBS as in Scheme B.

FIG. 81: (Scheme C) Use of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dGTP,3′-O-Biotin-SS-dCTP), 3′-O-SS(DTM)-dNTP-SS-Dye Clusters(3′-O-SS-dATP-7-SS-Rox Cluster, 3′-O-SS-dTTP-5-SS-Alexa488 Cluster), andappropriate dye labeled anchor binding molecules (Tetrazine-Rox Cluster,Streptavidin-Alexa488 Cluster) to perform 2-color DNA SBS. Step 1,addition of DNA polymerase and the four nucleotide analogues(3′-O-TCO-SS-dGTP, 3′-O-Biotin-SS-dCTP, 3′-O-SS-dATP-7-SS-Rox Clusterand 3′-O-SS-dTTP-5-SS-Alexa488 Cluster) to the immobilized primed DNAtemplate enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand to terminate DNA synthesis. Step 2,Chase: addition of DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue in case the primer was notextended with any of the dye or anchor labeled dNTPs in step 1. Thegrowing DNA strands are terminated with one of the four dye or anchorlabeled nucleotide analogues (A, C, G, T) or the equivalent nucleotideanalogues (A, C, G, T) without dye. In the case of single moleculesequencing, the base at this position in the growing DNA strand wouldnot be called, but because the 3′-OH will be restored in step 6,sequencing can still be carried out beyond this point. Step 3, afterwashing away the unincorporated dye labeled nucleotide analogues,detection of the fluorescence signal from the fluorescent dyes on theDNA products allows the identification of two of the incorporatednucleotide analogues for sequence determination. Rox signal indicatesincorporation of A, Alexa488 signal indicates incorporation of T. Step4, addition of Rox cluster-labeled tetrazine and Alexa488cluster-labeled streptavidin which bind to the TCO and biotin anchorsrespectively. Step 5, after washing away the excess labeling molecules,a second round of detection of any new fluorescence signal allows theidentification of the incorporation of the remaining two nucleotideanalogues for sequence determination. Appearance of a Rox signalindicates incorporation of G, appearance of an Alexa488 signal indicatesincorporation of C. Next, in Step 6, treatment of the DNA products withTHP cleaves the SS linker, leading to the removal of the remainingfluorescent dye and the regeneration of a free 3′-OH group on the DNAextension product. Although Scheme C is presented here as a singlemolecule SBS method, it can also be used for ensemble sequencing withoutany design changes. Structures of modified nucleotide analogues used inthis scheme are shown in FIG. 82 (1-3).

FIG. 82: FIG. 82-1 Structures of 3′-O-Anchor-SS(DTM)-dNTPs(3′-O-TCO-SS-dGTP and 3′-O-Biotin-SS-dCTP), 3′-O-DTM(SS)-dNTP-SS-DyeCluster (3′-O-SS-dATP-7-SS-Rox Cluster and 3′-O-SS-dTTP-5-SS-Alexa488Cluster) for 2-color DNA SBS as in Scheme C. FIG. 82-2: Structure of thecorresponding Dye Labeled Binding Molecules (Rox Cluster LabeledTetrazine) for 2-color DNA SBS as in Scheme C. FIG. 82-3: The structureof Alexa488 Cluster Labeled Streptavidin for 2-color DNA SBS as inScheme C.

FIG. 83: Scheme D Use of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dGTP,3′-O-Biotin-SS-dCTP), 3′-O-SS(DTM)-dNTP-SS(DTM)-Dye(3′-O-SS-dATP-7-SS-Rox), 3′-O-SS(DTM)-dNTP-SS-ET Cassette(3′-O-SS-dTTP-5-SS-[Rox---Cy5]) and appropriate dye labeled anchorbinding molecules (Streptavidin-Rox, Tetrazine-[Rox---Cy5]) to perform2-color DNA SBS. Step 1, addition of DNA polymerase and the fournucleotide analogues (3′-O-TCO-SS-dGTP, 3′-O-Biotin-SS-dCTP,3′-O-SS-dATP-7-SS-Rox and 3′-O-SS-dTTP-5-SS-[Rox---Cy5]) to theimmobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. Step 2, Chase: addition of DNA polymerase and four3′-O-SS(DTM)-dNTPs (3′-O-t-Butyldithiomethyl(SS)-dATP,3′-O-t-Butyldithiomethyl(SS)-dCTP, 3′-O-t-Butyldithiomethyl(SS)-dTTP and3′-O-t-Butyldithiomethyl(SS)-dGTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary3′-O-SS-nucleotide analogue to the subset of growing DNA strands in theensemble that were not extended with any of the dye or anchor labeleddNTPs in step 1. The growing DNA strands are terminated with one of thefour dye or anchor labeled nucleotide analogues (A, C, G, T) or theequivalent nucleotide analogues (A, C, G, T) without dye. Step 3, afterwashing away the unincorporated dye labeled nucleotide analogues,detection of the fluorescence signal from the fluorescent dyes on theDNA products allows the identification of two of the incorporatednucleotide analogues for sequence determination. Rox signal indicatesincorporation of A, Cy5 signal indicates incorporation of T. Step 4,addition of Rox-labeled streptavidin and [Rox . . . Cy5]cassette-labeled tetrazine which bind to the biotin and TCO anchorsrespectively. Step 5, after washing away the excess labeling molecules,a second round of detection of any new fluorescence signal allows theidentification of the incorporation of the remaining two nucleotideanalogues for sequence determination. Appearance of a Rox signalindicates incorporation of C, appearance of a Cy5 signal indicatesincorporation of G. Note that specific excitation of the donor dye, Rox,will result in emission of light at wavelengths that overlap theabsorbance spectrum of the acceptor dye, Cy5. As shown in FIG. 84, theposition of Rox and Cy5 on the polymeric molecule attached to the baseis chosen to produce optimal energy transfer. Next, in Step 6, treatmentof the DNA products with THP cleaves the SS linker, leading to theremoval of the remaining fluorescent dye and the regeneration of a free3′-OH group on the DNA extension product. Although Scheme D is presentedhere as an ensemble SBS approach, it can also be used for singlemolecule SBS sequencing with an appropriate imaging setup. Structures ofmodified nucleotide analogues used in this scheme are shown in FIG. 84.

FIG. 84: Structures of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dGTP and3′-O-Biotin-SS-dCTP), 3′-O-DTM(SS)-dNTP-SS-ET Cassette(3′-O-SS-dATP-7-SS-Rox and 3′-O-SS-dTTP-5-SS-Rox----Cy5 ET Cassette) andthe corresponding Dye Labeled Binding Molecules (Rox LabeledStreptavidin and Rox----Cy5 ET Cassette Labeled Tetrazine) for 2-colorDNA SBS as in Scheme D.

FIG. 85: (Scheme E) Use of 3′-O-SS(DTM)-dNTP-Azo-Anchors(3′-O-SS-dATP-7-Azo-TCO, 3′-O-SS-dCTP-5-Azo-Biotin);3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dGTP, 3′-O-Biotin-SS-dTTP) andappropriate dye labeled anchor binding molecule (Tetrazine-ATTO647N,Streptavidin-ATTO647N) to perform 1-color DNA SBS. Step 1, addition ofDNA polymerase and the four nucleotide analogues(3′-O-SS-dATP-7-Azo-TCO, 3′-O-SS-dCTP-5-Azo-Biotin, 3′-O-TCO-SS-dGTP,and 3′-O-Biotin-SS-dTTP) to the immobilized primed DNA template enablesthe incorporation of the complementary nucleotide analogue to thegrowing DNA strand to terminate DNA synthesis. Step 2, Chase: additionof DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the subset of growing DNAstrands in the ensemble that were not extended with any of the dye oranchor labeled dNTPs in step 1. The growing DNA strands are terminatedwith one of the four dye or anchor labeled nucleotide analogues (A, C,G, T) or the equivalent nucleotide analogues (A, C, G, T) without dye.Step 3, addition of ATTO647N-labeled streptavidin which binds tonucleotide analogues with biotin anchors. Step 4, after washing away theremaining labeling molecules, detection of a fluorescence signalindicates incorporation of either T or C. Step 5, addition ofATTO647N-labeled tetrazine which binds to nucleotide analogues with TCOanchors. Step 6, after washing away the excess labeling molecules,appearance of a previously absent fluorescence signal confirms theincorporation of either A or G. Step 7, treatment with sodium dithioniteto cleave the Azo linkers on A and C nucleotide analogues. Afterwashing, in Step 8 imaging is carried out a third time to detectremaining fluorescent signals. If we have already determined that theincorporated nucleotide could be T or C, loss of fluorescence wouldreveal it to be C, while remaining fluorescence would reveal it to be T.Similarly, for signals previously determined as A or G, loss offluorescence would indicate incorporation of A specifically whileremaining fluorescence would indicate incorporation of G. Next, in Step9, treatment of the DNA products with THP cleaves the SS linker, leadingto the removal of the remaining fluorescent dye and the regeneration ofa free 3′-OH group on the DNA extension product. Step 10, after washingaway the THP, optional imaging to confirm absence of any remainingfluorescent label indicates readiness for the next cycle of the DNAsequencing reaction. Although Scheme E is presented here as an ensembleSBS approach, it can also be used for single molecule SBS sequencingwith an appropriate imaging setup. Structures of modified nucleotideanalogues used in this scheme are shown in FIG. 86.

FIG. 86: 3′-O-DTM(SS)-dNTP-Azo-Anchors (3′-O-SS-dATP-7-Azo-TCO and3′-O-SS-dCTP-5-Azo-Biotin), 3′-O-Anchor-DTM(SS)-dNTP (3′-O-TCO-SS-dGTPand 3′-O-Biotin-SS-dTTP) and the dye labeled anchor binding molecule(ATTO647N labeled streptavidin) for 1-color DNA SBS as in Scheme E.

FIG. 87: (Scheme F) Use of 3′-O-SS(DTM)-dNTP-Azo-Anchor(3′-O-SS-dCTP-5-Azo-TCO), 3′-O-Anchor-SS(DTM)-dNTP (3′-O-TCO-SS-dGTP),3′-O-SS(DTM)-dNTP-Azo-Dye (3′-O-SS-dTTP-5-Azo-Rox),3′-O-Dye-SS(DTM)-dNTP (3′-O-Rox-SS-dATP) and appropriate dye labeledanchor binding molecule (Tetrazine-Rox) to perform 1-color DNA SBS. Step1, addition of DNA polymerase and the four nucleotide analogues(3′-O-SS-dCTP-5-Azo-TCO, 3′-O-TCO-SS-dGTP, 3′-O-SS-dTTP-5-Azo-Rox, and3′-O-Rox-SS-dATP) to the immobilized primed DNA template enables theincorporation of the complementary nucleotide analogue to the growingDNA strand to terminate DNA synthesis. Step 2, Chase: addition of DNApolymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the subset of growing DNAstrands in the ensemble that were not extended with any of the dye oranchor labeled dNTPs in step 1. The growing DNA strands are terminatedwith one of the four dye or anchor labeled nucleotide analogues (A, C,G, T) or the equivalent nucleotide analogues (A, C, G, T) without dye.Step 3, after washing away unincorporated nucleotide analogues, imagingis performed to detect fluorescence of incorporated nucleotideanalogues. Rox fluorescence indicates incorporation of either A or T.Step 4, addition of Rox-labeled tetrazine which binds to nucleotideanalogues with TCO anchors. Step 5, after washing away the remaininglabeling molecules, detection of a previously missing Rox signalconfirms incorporation of either C or G. Step 6, treatment with sodiumdithionite to cleave Azo linkers on T and C nucleotide analogues. Afterwashing, in Step 7 imaging is carried out a third time to detectremaining fluorescent signals. If we have already determined that theincorporated nucleotide could be A or T, loss of fluorescence wouldreveal it to be T, while remaining fluorescence would reveal it to be A.Similarly, for signals previously determined as C or G, loss offluorescence would indicate incorporation of C specifically whileremaining fluorescence would indicate incorporation of G. Next, in Step8, treatment of the DNA products with THP cleaves the SS linker, leadingto the removal of the remaining fluorescent dye and the regeneration ofa free 3′-OH group on the DNA extension product in readiness for thenext cycle of the DNA sequencing reaction. Although Scheme F ispresented here as an ensemble SBS approach, it can also be used forsingle molecule SBS sequencing with an appropriate imaging setup.Structures of modified nucleotide analogues used in this scheme areshown in FIG. 88.

FIG. 88: Structures of 3′-O-Dye-DTM(SS)-dNTPs (3′-O-Rox-SS-dATP),3′-O-DTM(SS)-dNTP-Azo-Dye (3′-O-SS-dTTP-5-Azo-Rox),3′-O-DTM(SS)-dNTP-Azo-Anchor (3′-O-SS-dCTP-5-Azo-TCO) and3′-O-Anchor-SS-dNTP (3′-O-TCO-SS-dGTP) as well as the dye labeledbinding molecule (Rox Labeled Tetrazine) for 1-color DNA SBS as inScheme F.

FIG. 89: Scheme G Use of 3′-O-SS(DTM)-dNTP-SS-Dye(3′-O-SS-dGTP-7-SS-ATTO647N), 3′-O-Anchor-SS(DTM)-dNTP(3′-O-Biotin-SS-dCTP), 3′-O-Anchor-Allyl-dNTP (3′-O-Biotin-Allyl-dATP)and 3′-O-Anchor-2NB-dNTP (3′-O-Biotin-2NB-dTTP) and appropriate dyelabeled anchor binding molecule (Streptavidin-ATTO647N) to perform1-color ensemble DNA SBS. Step 1, addition of DNA polymerase and thefour nucleotide analogues (3′-O-SS-dGTP-7-SS-ATTO647N,3′-O-Biotin-SS-dCTP, 3′-O-Biotin-Allyl-dATP, and 3′-O-Biotin-2NB-dTTP)to the immobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. Step 2, Chase: addition of DNA polymerase and four3′-O-SS(DTM)-dNTPs (3′-O-t-Butyldithiomethyl(SS)-dATP,3′-O-t-Butyldithiomethyl(SS)-dCTP, 3′-O-t-Butyldithiomethyl(SS)-dTTP and3′-O-t-Butyldithiomethyl(SS)-dGTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary3′-O-SS-nucleotide analogue to the subset of growing DNA strands in theensemble that were not extended with any of the dye or anchor labeleddNTPs in step 1. The growing DNA strands are terminated with one of thefour dye or anchor labeled nucleotide analogues (A, C, G. T) or theequivalent nucleotide analogues (A, C, G, T) without dye. Step 3, afterwashing away unincorporated nucleotide analogues, imaging is performedto detect fluorescence of incorporated nucleotide analogues. ATTO647Nfluorescence indicates incorporation of G. Step 4, addition ofATTO647N-labeled streptavidin which binds to nucleotide analogues withbiotin anchors. Step 5, after washing away the remaining labelingmolecules, an optional imaging step is carried out. Detection of a newATTO647N signal confirms incorporation of any one of A, C or T. Step 6,treatment with Pd(0) to cleave allyl linker on A. After washing, in Step7 imaging is carried out to detect remaining fluorescent signals. Lossof ATTO647N signal indicates an A was incorporated. Step 8, treatmentwith 340 nm light to cleave 2-nitrobenzyl linker on T. After washing, inStep 7 imaging is carried out to detect remaining fluorescent signals.Loss of ATTO647N signal indicates a T was incorporated. Remaining signalafter the two cleavage steps indicates incorporation of C, since a Gwould have been seen in the first imaging step. Next, in Step 9,treatment of the DNA products with THP cleaves the SS linker, leading tothe removal of the remaining fluorescent dye and the regeneration of afree 3′-OH group on the DNA extension product in readiness for the nextcycle of the DNA sequencing reaction. An optional imaging step showingabsence of fluorescence would confirm C incorporation. Although Scheme Gis presented here as an ensemble SBS approach, it can also be used forsingle molecule SBS sequencing with an appropriate imaging setup.Structures of modified nucleotide analogues used in this scheme areshown in FIG. 90.

FIG. 90: Structures of 3′-O-Anchor-CleavableLinker-dNTPs(3′-O-Biotin-Allyl-dATP, 3′-O-Biotin-SS-dCTP, 3′-O-Biotin-NB-dTTP),3′-O-DTM(SS)-dNTP-SS-Dye (3′-O-SS-dGTP-7-SS-ATTO647N) and thecorresponding Dye Labeled Binding Molecules (ATTO647N LabeledStreptavidin) for 1-color DNA SBS as in Scheme G.

FIG. 91: (Scheme H) Use of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dGTP,3′-O-Biotin-SS-dCTP), 3′-O-SS(DTM)-dNTP-SS-Dye Clusters(3′-O-SS-dATP-7-SS-Rox Cluster), 3′-O-SS(DTM)-dNTP-Azo-Dye Clusters(3′-O-SS-dTTP-5-Azo-Rox Cluster), and appropriate dye labeled anchorbinding molecules (Tetrazine-Rox Cluster, Streptavidin-Rox Cluster) toperform 1-color DNA SBS. Step 1, addition of DNA polymerase and the fournucleotide analogues (3′-O-TCO-SS-dGTP, 3′-O-Biotin-SS-dCTP,3′-O-SS-dATP-7-SS-Rox Cluster and 3′-O-SS-dTTP-5-Azo-Rox Cluster) to theimmobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. Step 2, Chase: addition of DNA polymerase and four3′-O-SS(DTM)-dNTPs (3′-O-t-Butyldithiomethyl(SS)-dATP,3′-O-t-Butyldithiomethyl(SS)-dCTP, 3′-O-t-Butyldithiomethyl(SS)-dTTP and3′-O-t-Butyldithiomethyl(SS)-dGTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary3′-O-SS-nucleotide analogue in case the primer was not extended with anyof the dye or anchor labeled dNTPs in step 1. The growing DNA strandsare terminated with one of the four dye or anchor labeled nucleotideanalogues (A, C, G, T) or the equivalent nucleotide analogues (A, C, G.T) without dye. In the case of single molecule sequencing, the base atthis position in the growing DNA strand would not be called, but becausethe 3′-OH will be restored in step 10, sequencing can still be carriedout beyond this point. Step 3, after washing away the unincorporated dyelabeled nucleotide analogues, detection of the fluorescence signal fromthe fluorescent dyes on the DNA products allows the identification ofeither of two of the incorporated nucleotide analogues for sequencedetermination. Rox signal indicates incorporation of A or T. Step 4,addition of Rox cluster-labeled streptavidin to bind to the biotinanchors. Step 5, after washing away the excess labeling molecules, asecond round of detection is performed. Appearance of a new Rox signalconfirms incorporation of C. Step 6, addition of Rox cluster-labeledtetrazine to bind to the TCO anchors. Step 7, after washing away theexcess labeling molecules, a third round of detection is performed.Appearance of a new Rox signal confirms incorporation of G. Step 8,treatment with sodium dithionite cleaves the Azo linkers on T nucleotideanalogues. Step 9, after washing, loss of Rox signal indicatesincorporation of T; remaining Rox signal indicates incorporation of A.Finally, in Step 10, treatment of the DNA products with THP cleaves theDTM linker, removing the fluorescent dye from the T nucleotide analogueand regenerating a free 3′-OH group on the DNA extension product. Atthis point, the the DNA is ready for the next sequencing cycle. AlthoughScheme H is presented here as a single molecule SBS methods, it can alsobe used for ensemble sequencing without any design changes. Structuresof modified nucleotide analogues used in this scheme are shown in FIGS.92-1 and 92-2).

FIG. 92: FIG. 92-1: Structures of 3′-O-Anchor-SS(DTM)-dNTPs(3′-O-TCO-SS-dGTP and 3′-O-Biotin-SS-dCTP), 3′-O-DTM(SS)-dNTP-SS-DyeCluster (3′-O-SS-dATP-7-SS-Rox Cluster), 3′-O-DTM(SS)-dNTP-Azo-DyeCluster (3′-O-SS-dTTP-5-Azo-Rox Cluster) and the corresponding DyeLabeled Binding Molecules (Rox Cluster Labeled Streptavidin (FIG. 92-2)and Rox Cluster Labeled Tetrazine (FIG. 82-2)) for 1-color DNA SBS as inScheme H. FIG. 92-2: The structure of Rox Cluster Labeled Streptavidinfor 1-color DNA SBS as in Scheme H.

FIG. 93: Synthesis of 3′-O-SS-dTTP-SS-(Rox--Cy5 ET Cassette). TheAlkynyl-Rox-Cy5 ET Cassette can be routinely synthesized by using thestandard oligonucleotide synthesis approach.

FIG. 94: General synthesis of 3′-SS-dNTP-Azo-Dye Cluster (dATP as anexample).

FIG. 95: Synthesis of 3′-O-SS-dTTP-Azo-(Rox Cluster). The Alkynyl-RoxCluster can be routinely synthesized by using the standardoligonucleotide synthesis approach.

FIG. 96: Synthesis of 3′-O-SS-dATP-SS-(Rox Cluster). The Alkynyl-RoxCluster can be routinely synthesized by using the standardoligonucleotide synthesis approach.

FIG. 97: Synthesis of Rox Cluster labeled tetrazine. The 5′-Amino-RoxCluster can be routinely synthesized by using the standardoligonucleotide synthesis approach.

FIG. 98: General Synthesis of 3′-O-SS-dNTP-Azo-Dye (Anchor).

FIG. 99: Synthesis of 3′-O-SS-dTTP-5-Azo-BodipyFL and3′-O-SS-dGTP-7-Azo-Rox.

FIG. 100: Synthesis of 3′-O-SS-dCTP-5-Azo-Biotin and3′-O-SS-dATP-7-Azo-TCO.

DETAILED DESCRIPTION

This invention provides novel nucleotide analogs containing a3′-O-labeled reversibly removable moiety that are efficientlyincorporated by DNA polymerases into the growing DNA strand totemporarily terminate the reaction and produce a DNA extension productcarrying the fluorescent label. By detecting the signal from thefluorophore, the identity of the incorporated nucleotide is determined(e.g., by the process of sequencing by synthesis (SBS)). Then theDye-DTM moiety on the 3′ of the DNA extension product is removed bytreatment with Tris(3-hydroxypropyl)phosphine (THP) in an aqueous buffersolution to regenerate the 3′-OH group, which allows the re-initiationof the polymerase reaction for incorporation of the next incoming3′-O-Dye-DTM-dNTP with high efficiency. Consecutive SBS using3′-O-Dye-DTM-dNTP as reversible terminator generates a natural DNAstrand, allowing the generation of accurate DNA sequencing data withlong read length.

This invention provides novel nucleotide analogues containing a3′-O-modification that can be efficiently incorporated by DNApolymerases into the growing DNA strand to temporarily terminate thereaction and produce a DNA extension product carrying a detectablelabel. The invention further provides novel nucleotide analoguescomprising a 3′-O-labeled reversibly removable moiety and an anchormoiety, which is a predetermined small chemical group correlated to theidentity of the base and that orthogonally and rapidly reacts with acomplementary binding molecule thereby joining the anchor and bindingmolecule so as to form a conjugate. The complementary binding moleculecomprises a detectable label and a binder that binds to the anchor onthe nucleotide and a detectable label. By detecting the signal from thedetectable label, whether attached to an incorporated nucleotideanalogue, or attached to a binding molecule that has formed a conjugatewith a nucleotide analogue, the identity of the incorporated nucleotideis determined. Then the 3′-O moiety of the DNA extension product isremoved by treatment with a water soluble phosphine in an aqueous buffersolution to regenerate the 3′-OH group, which allows the re-initiationof the polymerase reaction for incorporation of the next incomingnucleotide analogue. The use of the following nucleotide analogues invarious combinations to perform SBS are described: (a) those withfluorophores attached at the 3′-O position via a cleavable linker, (b)those with cleavable anchors at the 3′-O position for subsequentattachment of fluorophores, and (c) those with cleavable fluorophores onthe base and a reversible blocking group on the 3′-OH. Consecutive SBSusing the disclosed nucleotide analogues as reversible terminatorsgenerates a natural DNA strand, allowing the generation of accurate DNAsequencing data with long read length.

I. Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂-.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Alkyl is an uncyclized chain.Examples of saturated hydrocarbon radicals include, but are not limitedto, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, sec-butyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. An alkoxy is an alkyl attached to theremainder of the molecule via an oxygen linker (—O—). An alkyl moietymay be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. Analkyl moiety may be fully saturated. An alkenyl may include more thanone double bond and/or one or more triple bonds in addition to the oneor more double bonds. An alkynyl may include more than one triple bondand/or one or more double bonds in addition to the one or more triplebonds.

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂-. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred herein. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkelyene (e.g.,alkylene, alkenylene, or alkynylene) group, generally having eight orfewer carbon atoms. The term “alkenylene,” by itself or as part ofanother substituent, means, unless otherwise stated, a divalent radicalderived from an alkene. The term “alkynylene” by itself or as part ofanother substituent, means, unless otherwise stated, a divalent radicalderived from an alkyne.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom (e.g., O, N, P, Si, B, and S), and wherein the nitrogenand sulfur atoms may optionally be oxidized, and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, B,or P) may be placed at any interior position of the heteroalkyl group orat the position at which the alkyl group is attached to the remainder ofthe molecule. Heteroalkyl is an uncyclized chain. Examples include, butare not limited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and —CN. Up to two or threeheteroatoms may be consecutive, such as, for example, —CH₂-NH-OCH₃ and—CH₂-O-Si(CH₃)₃. A heteroalkyl moiety may include one heteroatom (e.g.,O, N, S, Si, B, or P). A heteroalkyl moiety may include two optionallydifferent heteroatoms (e.g., O, N, S, Si, B, or P). A heteroalkyl moietymay include three optionally different heteroatoms (e.g., O, N, S, Si,B, or P). A heteroalkyl moiety may include four optionally differentheteroatoms (e.g., O, N, S, Si, B, or P). A heteroalkyl moiety mayinclude five optionally different heteroatoms (e.g., O, N, S, Si, B, orP). A heteroalkyl moiety may include up to 8 optionally differentheteroatoms (e.g., O, N, S, Si, B, or P). The term “heteroalkenyl,” byitself or in combination with another term, means, unless otherwisestated, a heteroalkyl including at least one double bond. Aheteroalkenyl may optionally include more than one double bond and/orone or more triple bonds in additional to the one or more double bonds.The term “heteroalkynyl” by itself or in combination with another term,means, unless otherwise stated, a heteroalkyl including at least onetriple bond. A heteroalkynyl may optionally include more than one triplebond and/or one or more double bonds in additional to the one or moretriple bonds.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkelyenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkelyene (e.g., alkylene, alkenylene, oralkynylene) and heteroalkelyene linking groups, no orientation of thelinking group is implied by the direction in which the formula of thelinking group is written. For example, the formula —C(O)₂R′— representsboth —C(O)₂R′— and —R′C(O)₂—. As described above, heteroalkyl groups, asused herein, include those groups that are attached to the remainder ofthe molecule through a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″,—OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” is recited, followed byrecitations of specific heteroalkyl groups, such as-NR′R″ or the like,it will be understood that the terms heteroalkyl and —NR′R″ are notredundant or mutually exclusive. Rather, the specific heteroalkyl groupsare recited to add clarity. Thus, the term “heteroalkyl” should not beinterpreted herein as excluding specific heteroalkyl groups, suchas-NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl andheterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl,pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl,oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl,benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl,indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl,quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl,3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl,2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively. Aheteroaryl group substituent may be —O— bonded to a ring heteroatomnitrogen.

Spirocyclic rings are two or more rings wherein adjacent rings areattached through a single atom. The individual rings within spirocyclicrings may be identical or different. Individual rings in spirocyclicrings may be substituted or unsubstituted and may have differentsubstituents from other individual rings within a set of spirocyclicrings. Possible substituents for individual rings within spirocyclicrings are the possible substituents for the same ring when not part ofspirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkylrings). Spirocylic rings may be substituted or unsubstituted cycloalkyl,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heterocycloalkelyeneand individual rings within a spirocyclic ring group may be any of theimmediately previous list, including having all rings of one type (e.g.all rings being substituted heterocycloalkelyene wherein each ring maybe the same or different substituted heterocycloalkylene). Whenreferring to a spirocyclic ring system, heterocyclic spirocyclic ringsmeans a spirocyclic rings wherein at least one ring is a heterocyclicring and wherein each ring may be a different ring. When referring to aspirocyclic ring system, substituted spirocyclic rings means that atleast one ring is substituted and each substituent may optionally bedifferent.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylarylene” as an arylene moiety covalently bonded to analkelyene (e.g., alkylene, alkenylene, or alkynylene) moiety (alsoreferred to herein as an alkelyene). In embodiments, the alkylarylenegroup has the formula:

An alkylarylene moiety may be substituted (e.g., with a substituentgroup) on the alkelyene (e.g., alkylene, alkenylene, or alkynylene)moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) withhalogen, oxo, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —CHO, —OH, —NH₂,—COOH,-CONH₂, —NO₂, —SH, —SO₂CH₃—SO₃H, —OSO₃H, —SO₂NH₂, NHNH₂, ONH₂,NHC(O)NHNH₂, substituted or unsubstituted C₁-C₅ alkyl or substituted orunsubstituted 2 to 5 membered heteroalkyl). In embodiments, thealkylarylene is unsubstituted.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,”“heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substitutedand unsubstituted forms of the indicated radical. Preferred substituentsfor each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R″′, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R″′)═NR″″,—NR—C(NR′R″)═NR″′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, NR′NR″R″′,ONR′R″, NR′C(O)NR′NR″′R″′, —CN, —NO₂, —NR′SO₂R″, —NR′C(O)R″,—NR′C(O)—OR″, —NR′OR″, in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R, R′, R″, R″′,and R″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When acompound described herein includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ group when more than one of these groups is present. When R′ andR″ are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R″′, —NR″C(O)₂R′, —NR—C(NR′R″R″′)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, NR′NR″R″′, ONR′R″,NR′C(O)NR″NR″′R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, —NR′SO₂R″, —NR′C(O)R″, —NR′C(O)—OR″, —NR′OR″, ina number ranging from zero to the total number of open valences on thearomatic ring system; and where R′, R″, R″′, and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. When a compound described herein includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R″′, and R″″ groups when more than one of these groupsis present.

Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl,heteroaryl, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene) may be depicted as substituents on the ring rather thanon a specific atom of a ring (commonly referred to as a floatingsubstituent). In such a case, the substituent may be attached to any ofthe ring atoms (obeying the rules of chemical valency) and in the caseof fused rings or spirocyclic rings, a substituent depicted asassociated with one member of the fused rings or spirocyclic rings (afloating substituent on a single ring), may be a substituent on any ofthe fused rings or spirocyclic rings (a floating substituent on multiplerings). When a substituent is attached to a ring, but not a specificatom (a floating substituent), and a subscript for the substituent is aninteger greater than one, the multiple substituents may be on the sameatom, same ring, different atoms, different fused rings, differentspirocyclic rings, and each substituent may optionally be different.Where a point of attachment of a ring to the remainder of a molecule isnot limited to a single atom (a floating substituent), the attachmentpoint may be any atom of the ring and in the case of a fused ring orspirocyclic ring, any atom of any of the fused rings or spirocyclicrings while obeying the rules of chemical valency. Where a ring, fusedrings, or spirocyclic rings contain one or more ring heteroatoms and thering, fused rings, or spirocyclic rings are shown with one more floatingsubstituents (including, but not limited to, points of attachment to theremainder of the molecule), the floating substituents may be bonded tothe heteroatoms. Where the ring heteroatoms are shown bound to one ormore hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and athird bond to a hydrogen) in the structure or formula with the floatingsubstituent, when the heteroatom is bonded to the floating substituent,the substituent will be understood to replace the hydrogen, whileobeying the rules of chemical valency.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)-X′—(C″R″R″′)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R″′ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P),and silicon (Si).

A “substituent” or “substituent group,” as used herein, means a groupselected from the following moieties:

(A) oxo, halogen, —CF, —CHF₂, —CH₂F, —C(halogen)₃, —CH(halogen)₂,—CH₂(halogen), —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, NHNH₂, ONH₂, NHC(O)NHNH₂, NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCHF₂, —OCH₂F, —OCF₃, —OCHF₂, —OCH₂F,—OC(halogen). —OCH(halogen)₂, —OCH₂(halogen), unsubstituted alkyl (e.g.,C₁-C₂₀, C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), unsubstituted heteroalkyl (e.g.,2 to 20 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2to 3 membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3to 8 membered, 3 to 6 membered. 4 to 6 membered, 4 to 5 membered, or 5to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀ or phenyl),unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered); and(B) alkyl (e.g., C₁-C₂₀, C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), heteroalkyl(e.g., 2 to 20 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6membered, 2 to 3 membered, or 4 to 5 membered), cycloalkyl (e.g., C₃-C₈,C₃-C₆, C₄-C₆, or C₅-C₆), heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), aryl(e.g., C₆-C₁₀ or phenyl), heteroaryl (e.g., 5 to 10 membered, 5 to 9membered, or 5 to 6 membered), substituted with at least one substituentselected from:

-   -   (i) oxo, halogen, —CF₃, —CHF₂, —CH₂F, —C(halogen)₃,        —CH(halogen)₂, —CH₂(halogen), —CN, —OH, —NH₂, —COOH, —CONH₂,        —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,        —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCHF₂,        —OCH₂F, —OCF₃, —OCHF₂, —OCH₂F, —OC(halogen)₃, —OCH(halogen)₂,        —OCH₂(halogen), unsubstituted alkyl (e.g., C₁-C₂₀, C₁-C₈, C₁-C₆,        C₁-C₄, or C₁-C₂), unsubstituted heteroalkyl (e.g., 2 to 20        membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2        to 3 membered, or 4 to 5 membered), unsubstituted cycloalkyl        (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), unsubstituted        heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6        membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted        aryl (e.g., C₆-C₁₀ or phenyl), unsubstituted heteroaryl (e.g., 5        to 10 membered, 5 to 9 membered, or 5 to 6 membered), and    -   (ii) alkyl (e.g., C₁-C₂₀, C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂),        heteroalkyl (e.g., 2 to 20 membered, 2 to 8 membered, 2 to 6        membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),        cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),        heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6        membered, 4 to 5 membered, or 5 to 6 membered), aryl (e.g.,        C₆-C₁₀ or phenyl), heteroaryl (e.g., 5 to 10 membered, 5 to 9        membered, or 5 to 6 membered), substituted with at least one        substituent selected from:        -   (a) oxo, halogen, —CF₃, —CHF₂, —CH₂F, —C(halogen)₃,            —CH(halogen)₂, —CH₂(halogen), —CN, —OH, —NH₂, —COOH, —CONH₂,            —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, NHNH₂, ONH₂, NHC(O)NHNH₂,            NHC(O)NH₂,—NHSO₂H, —NHC(O)H, NHC(O)OH, —NHOH, —OCF₃, —OCHF₂,            —OCH₂F, —OCF₃, —OCHF₂, —OCH₂F, —OC(halogen)₃,            —OCH(halogen)₂, —OCH₂(halogen), unsubstituted alkyl (e.g.,            C₁-C₂₀, C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), unsubstituted            heteroalkyl (e.g., 2 to 20 membered, 2 to 8 membered, 2 to 6            membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5            membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,            C₄-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to            8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5            membered, or 5 to 6 membered), unsubstituted aryl (e.g.,            C₆-C₁₀ or phenyl), unsubstituted heteroaryl (e.g., 5 to 10            membered, 5 to 9 membered, or 5 to 6 membered), and        -   (b) alkyl (e.g., C₁-C₂₀, C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂),            heteroalkyl (e.g., 2 to 20 membered, 2 to 8 membered, 2 to 6            membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5            membered), cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),            heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4            to 6 membered, 4 to 5 membered, or 5 to 6 membered), aryl            (e.g., C₆-C₁₀ or phenyl), heteroaryl (e.g., 5 to 10            membered, 5 to 9 membered, or 5 to 6 membered), substituted            with at least one substituent selected from:    -   oxo, halogen, —CF₃, —CHF₂. —CH₂F, —C(halogen)₃, —CH(halogen)₂,        —CH₂(halogen), —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,        —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂,        —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCHF₂, —OCH₂F,        —OCF₃, —OCHF₂, —OCH₂F, —OC(halogen)₃, —OCH(halogen)₂,        —OCH₂(halogen), unsubstituted alkyl (e.g., C₁-C₂₀, C₁-C₈, C₁-C₆,        C₁-C₄, or C₁-C₂), unsubstituted heteroalkyl (e.g., 2 to 20        membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2        to 3 membered, or 4 to 5 membered), unsubstituted cycloalkyl        (e.g., C₃-C₈, C₃-C₆, C₁-C₆, or C₅-C₆), unsubstituted        heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6        membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted        aryl (e.g., C₆-C₁₀ or phenyl), unsubstituted heteroaryl (e.g., 5        to 10 membered, 5 to 9 membered, or 5 to 6 membered).

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkelyene (e.g., alkylene, alkenylene, or alkynylene) is a substitutedor unsubstituted C₁-C₂₀ alkylene, each substituted or unsubstitutedheteroalkelyene is a substituted or unsubstituted 2 to 20 memberedheteroalkylene, each substituted or unsubstituted cycloalkelyene is asubstituted or unsubstituted C₃-C₈ cycloalkylene, each substituted orunsubstituted heterocycloalkelyene is a substituted or unsubstituted 3to 8 membered heterocycloalkylene, each substituted or unsubstitutedarylene is a substituted or unsubstituted C₆-C₁₀ arylene, and/or eachsubstituted or unsubstituted heteroarylene is a substituted orunsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkelyene (e.g., alkylene, alkenylene, or alkynylene) is asubstituted or unsubstituted C₁-C₈ alkylene, each substituted orunsubstituted heteroalkelyene is a substituted or unsubstituted 2 to 8membered heteroalkylene, each substituted or unsubstitutedcycloalkelyene is a substituted or unsubstituted C₃-C₇ cycloalkylene,each substituted or unsubstituted heterocycloalkelyene is a substitutedor unsubstituted 3 to 7 membered heterocycloalkylene, each substitutedor unsubstituted arylene is a substituted or unsubstituted C₆-C₁₀arylene, and/or each substituted or unsubstituted heteroarylene is asubstituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section, figures, or tables below.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those that are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

It should be noted that throughout the application that alternatives arewritten in Markush groups, for example, each amino acid position thatcontains more than one possible amino acid. It is specificallycontemplated that each member of the Markush group should be consideredseparately, thereby comprising another embodiment, and the Markush groupis not to be read as a single unit.

“Analog,” or “analogue” is used in accordance with its plain ordinarymeaning within Chemistry and Biology and refers to a chemical compoundthat is structurally similar to another compound (i.e., a so-called“reference” compound) but differs in composition, e.g., in thereplacement of one atom by an atom of a different element, or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group, or the absolutestereochemistry of one or more chiral centers of the reference compound.Accordingly, an analog is a compound that is similar or comparable infunction and appearance but not in structure or origin to a referencecompound.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls.

Moreover, where a moiety is substituted with an R substituent, the groupmay be referred to as “R-substituted.” Where a moiety is R-substituted,the moiety is substituted with at least one R substituent and each Rsubstituent is optionally different. Where a particular R group ispresent in the description of a chemical genus (such as Formula (I)), aRoman alphabetic symbol may be used to distinguish each appearance ofthat particular R group. For example, where multiple R¹³ substituentsare present, each R¹³ substituent may be distinguished as R^(13A),R^(13B), R^(13C), R^(13D), etc., wherein each of R^(13A), R^(13B),R^(13C), R^(13D), etc. is defined within the scope of the definition ofR¹³ and optionally differently.

A “detectable agent” or “detectable compound” or “detectable label” or“detectable moiety” is a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, chemical, magnetic resonanceimaging, or other physical means. For example, detectable agents include¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga,⁷⁷As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁴Tc, ⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh,¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm,¹⁵⁴⁻¹⁵⁸¹Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁵Lu, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra,²²⁵Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb,Dy, Ho, Er, Tm, Yb, Lu, ³²P, fluorophore (e.g. fluorescent dyes),modified oligonucleotides (e.g., moieties described inPCT/US2015/022063, which is incorporated herein by reference),electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles,ultrasmall superparamagnetic iron oxide (“USPIO”) nanoparticles, USPIOnanoparticle aggregates, superparamagnetic iron oxide (“SPIO”)nanoparticles, SPIO nanoparticle aggregates, monochrystalline iron oxidenanoparticles, monochrystalline iron oxide, nanoparticle contrastagents, liposomes or other delivery vehicles containing Gadoliniumchelate (“Gd-chelate”) molecules, Gadolinium, radioisotopes,radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18,rubidium-82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gammaray emitting radionuclides, positron-emitting radionuclide, radiolabeledglucose, radiolabeled water, radiolabeled ammonia, biocolloids,microbubbles (e.g. including microbubble shells including albumin.galactose, lipid, and/or polymers; microbubble gas core including air,heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexanelipid microsphere, perflutren, etc.), iodinated contrast agents (e.g.iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide,diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide,gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores,two-photon fluorophores, or haptens and proteins or other entities whichcan be made detectable, e.g., by incorporating a radiolabel into apeptide or antibody specifically reactive with a target peptide.

Radioactive substances (e.g., radioisotopes) that may be used asdetectable, imaging and/or labeling agents in accordance with theembodiments described herein include, but are not limited to, ¹⁸F, ³²P,³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷As, ⁸⁶Y,⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁹Tc, ⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In,¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸¹Gd, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra and ²²⁵Ac. Paramagneticions that may be used as additional imaging agents in accordance withthe embodiments of the disclosure include, but are not limited to, ionsof transition and lanthanide metals (e.g., metals having atomic numbersof 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn,Fe, Co, Ni, Cu. La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yband Lu.

Examples of detectable agents include imaging agents, includingfluorescent and luminescent substances, including, but not limited to, avariety of organic or inorganic small molecules commonly referred to as“dyes,” “labels,” or “indicators.” Examples include fluorescein,rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. In embodiments,the detectable moiety is a fluorescent molecule (e.g., acridine dye,cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, orrhodamine dye). In embodiments, the detectable moiety is a fluorescentmolecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye,phenanthridine dye, or rhodamine dye). In embodiments, the detectablemoiety is a fluorescein isothiocyanate moiety,tetramethylrhodamine-5-(and 6)-isothiocyanate moiety, Cy2 moeity, Cy3moiety, Cy5 moiety, Cy7 moiety, 4′,6-diamidino-2-phenylindole moiety,Hoechst 33258 moiety, Hoechst 33342 moiety, Hoechst 34580 moiety,propidium-iodide moiety, or acridine orange moiety. In embodiments, thedetectable moiety is a Indo-1, Ca saturated moiety, Indo-1 Ca2+ moiety,Cascade Blue BSA pH 7.0 moiety, Cascade Blue moiety, LysoTracker Bluemoiety, Alexa 405 moiety. LysoSensor Blue pH 5.0 moiety. LysoSensor Bluemoiety, DyLight 405 moiety, DyLight 350 moiety, BFP (Blue FluorescentProtein) moiety, Alexa 350 moiety, 7-Amino-4-methylcoumarin pH 7.0moiety, Amino Coumarin moiety, AMCA conjugate moiety, Coumarin moiety,7-Hydroxy-4-methylcoumarin moiety, 7-Hydroxy-4-methylcoumarin pH 9.0moiety, 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0 moiety, Hoechst33342 moiety, Pacific Blue moiety, Hoechst 33258 moiety, Hoechst33258-DNA moiety, Pacific Blue antibody conjugate pH 8.0 moiety,PO-PRO-1 moiety, PO-PRO-1-DNA moiety, POPO-1 moiety, POPO-1-DNA moiety,DAPI-DNA moiety, DAPI moiety. Marina Blue moiety, SYTOX Blue-DNA moiety,CFP (Cyan Fluorescent Protein) moiety, eCFP (Enhanced Cyan FluorescentProtein) moiety, 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS) moiety,Indo-1, Ca free moiety, 1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid)moiety, BO-PRO-1-DNA moiety, BOPRO-1 moiety, BOBO-1-DNA moiety. SYTO45-DNA moiety, evoglow-Pp1 moiety, evoglow-Bs1 moiety, evoglow-Bs2moiety, Auramine O moiety, DiO moiety, LysoSensor Green pH 5.0 moiety,Cy 2 moiety, LysoSensor Green moiety, Fura-2, high Ca moiety, Fura-2Ca2+sup>moiety, SYTO 13-DNA moiety, YO-PRO-1-DNA moiety, YOYO-1-DNAmoiety, eGFP (Enhanced Green Fluorescent Protein) moiety, LysoTrackerGreen moiety, GFP (S65T) moiety, BODIPY FL, MeOH moiety, Sapphiremoiety, BODIPY FL conjugate moiety, MitoTracker Green moiety,MitoTracker Green FM, MeOH moiety, Fluorescein 0.1 M NaOH moiety,Calcein pH 9.0 moiety. Fluorescein pH 9.0 moiety, Calcein moiety,Fura-2, no Ca moiety, Fluo-4 moiety, FDA moiety, DTAF moiety,Fluorescein moiety, CFDA moiety, FITC moiety, Alexa Fluor 488hydrazide-water moiety, DyLight 488 moiety, 5-FAM pH 9.0 moiety, Alexa488 moiety, Rhodamine 110 moiety, Rhodamine 110 pH 7.0 moiety, AcridineOrange moiety, BCECF pH 5.5 moiety. PicoGreendsDNA quantitation reagentmoiety, SYBR Green I moiety, Rhodaminen Green pH 7.0 moiety, CyQUANTGR-DNA moiety, NeuroTrace 500/525, green fluorescent Nissl stain-RNAmoiety. DansylCadaverine moiety, Fluoro-Emerald moiety, Nissl moiety,Fluorescein dextran pH 8.0 moiety, Rhodamine Green moiety,5-(and-6)-Carboxy-2′, 7′-dichlorofluorescein pH 9.0 moiety,DansylCadaverine, MeOH moiety, eYFP (Enhanced Yellow FluorescentProtein) moiety, Oregon Green 488 moiety, Fluo-3 moiety, BCECF pH 9.0moiety, SBFI-Na+ moiety, Fluo-3 Ca2+ moiety. Rhodamine 123 MeOH moiety,FlAsH moiety, Calcium Green-1 Ca2+ moiety, Magnesium Green moiety,DM-NERF pH 4.0 moiety, Calcium Green moiety, Citrine moiety, LysoSensorYellow pH 9.0 moiety, TO-PRO-1-DNA moiety, Magnesium Green Mg2+ moiety,Sodium Green Na+ moiety, TOTO-1-DNA moiety, Oregon Green 514 moiety,Oregon Green 514 antibody conjugate pH 8.0 moiety, NBD-X moiety, DM-NERFpH 7.0 moiety, NBD-X, MeOH moiety, CI-NERF pH 6.0 moiety, Alexa 430moiety, CI-NERF pH 2.5 moiety, Lucifer Yellow, CH moiety, LysoSensorYellow pH 3.0 moiety, 6-TET, SE pH 9.0 moiety. Eosin antibody conjugatepH 8.0 moiety, Eosin moiety, 6-Carboxyrhodamine 6G pH 7.0 moiety,6-Carboxyrhodamine 6G, hydrochloride moiety, Bodipy R6G SE moiety,BODIPY R6G MeOH moiety, 6 JOE moiety, Cascade Yellow moiety, mBananamoiety, Alexa 532 moiety, Erythrosin-5-isothiocyanate pH 9.0 moiety,6-HEX, SE pH 9.0 moiety, mOrange moiety, mHoneydew moiety, Cy 3 moiety,Rhodamine B moiety, DiI moiety, 5-TAMRA-MeOH moiety, Alexa 555 moiety,DyLight 549 moiety, BODIPY TMR-X, SE moiety, BODIPY TMR-X MeOH moiety,PO-PRO-3-DNA moiety, PO-PRO-3 moiety, Rhodamine moiety, POPO-3 moiety,Alexa 546 moiety, Calcium Orange Ca2+ moiety, TRITC moiety, CalciumOrange moiety, Rhodaminephalloidin pH 7.0 moiety, MitoTracker Orangemoiety, MitoTracker Orange MeOH moiety, Phycoerythrin moiety, MagnesiumOrange moiety, R-Phycoerythrin pH 7.5 moiety, 5-TAMRA pH 7.0 moiety,5-TAMRA moiety. Rhod-2 moiety, FM 1-43 moiety, Rhod-2 Ca2+ moiety, FM1-43 lipid moiety, LOLO-1-DNA moiety, dTomato moiety, DsRed moiety,Dapoxyl (2-aminoethyl) sulfonamide moiety, Tetramethylrhodamine dextranpH 7.0 moiety, Fluor-Ruby moiety, Resorufin moiety, Resorufin pH 9.0moiety, mTangerine moiety, LysoTracker Red moiety, Lissaminerhodaminemoiety, Cy 3.5 moiety, Rhodamine Red-X antibody conjugate pH 8.0 moiety,Sulforhodamine 101 EtOH moiety, JC-1 pH 8.2 moiety, JC-1 moiety,mStrawberry moiety, MitoTracker Red moiety, MitoTracker Red, MeOHmoiety. X-Rhod-1 Ca2+ moiety, Alexa 568 moiety, 5-ROX pH 7.0 moiety,5-ROX (5-Carboxy-X-rhodamine, triethylammonium salt) moiety,BO-PRO-3-DNA moiety, BOPRO-3 moiety, BOBO-3-DNA moiety, Ethidium Bromidemoiety, ReAsH moiety, Calcium Crimson moiety, Calcium Crimson Ca2+moiety, mRFP moiety, mCherry moiety, HcRed moiety, DyLight 594 moiety,Ethidium homodimer-1-DNA moiety, Ethidiumhomodimer moiety, PropidiumIodide moiety, SYPRO Ruby moiety, Propidium lodide-DNA moiety, Alexa 594moiety. BODIPY TR-X, SE moiety, BODIPY TR-X, MeOH moiety, BODIPY TR-Xphallacidin pH 7.0 moiety, Alexa Fluor 610 R-phycoerythrin streptavidinpH 7.2 moiety, YO-PRO-3-DNA moiety, Di-8 ANEPPS moiety,Di-8-ANEPPS-lipid moiety, YOYO-3-DNA moiety, Nile Red-lipid moiety, NileRed moiety, DyLight 633 moiety, mPlum moiety, TO-PRO-3-DNA moiety, DDAOpH 9.0 moiety, Fura Red high Ca moiety, Allophycocyanin pH 7.5 moiety,APC (allophycocyanin) moiety, Nile Blue, EtOH moiety, TOTO-3-DNA moiety,Cy 5 moiety, BODIPY 650/665-X, MeOH moiety, Alexa Fluor 647R-phycoerythrin streptavidin pH 7.2 moiety, DyLight 649 moiety, Alexa647 moiety, Fura Red Ca2+ moiety, Atto 647 moiety. Fura Red, low Camoiety, Carboxynaphthofluorescein pH 10.0 moiety, Alexa 660 moiety, Cy5.5 moiety, Alexa 680 moiety, DyLight 680 moiety, Alexa 700 moiety, FM4-64, 2% CHAPS moiety, or FM 4-64 moiety. In embodiments, the dectablemoiety is a moiety of 1,1-Diethyl-4,4-carbocyanine iodide,1,2-Diphenylacetylene, 1,4-Diphenylbutadiene, 1,4-Diphenylbutadiyne,1,6-Diphenylhexatriene, 1,6-Diphenylhexatriene,1-anilinonaphthalene-8-sulfonic acid, 2,7-Dichlorofluorescein,2,5-DIPHENYLOXAZOLE, 2-Di-1-ASP, 2-dodecylresorufin,2-Methylbenzoxazole, 3,3-Diethylthiadicarbocyanine iodide,4-Dimethylamino-4-Nitrostilbene, 5(6)-Carboxyfluorescein,5(6)-Carboxynaphtofluorescein 5(6)-Carboxytetramethylrhodamine B,5-(and-6)-carboxy-2′,7′-dichlorofluorescein,5-(and-6)-carboxy-2,7-dichlorofluorescein, 5-(N-hexadecanoyl)aminoeosin,5-(N-hexadecanoyl)aminoeosin, 5-chloromethylfluorescein, 5-FAM, 5-ROX,5-TAMRA, 5-TAMRA, 6,8-difluoro-7-hydroxy-4-methylcoumarin,6,8-difluoro-7-hydroxy-4-methylcoumarin, 6-carboxyrhodamine 6G, 6-HEX,6-JOE, 6-JOE, 6-TET, 7-aminoactinomycin D,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, 7-Methoxycoumarin-4-AceticAcid, 8-Benzyloxy-5,7-diphenylquinoline,8-Benzyloxy-5,7-diphenylquinoline, 9,10-Bis(Phenylethynyl)Anthracene,9,10-Diphenylanthracene, 9-METHYLCARBAZOLE, (CS)2Ir(μ-Cl)2Ir(CS)2, AAA,Acridine Orange, Acridine Orange, Acridine Yellow, Acridine Yellow,Adams Apple Red 680, Adirondack Green 520, Alexa Fluor 350, Alexa Fluor405, Alexa Fluor 430, Alexa Fluor 430, Alexa Fluor 480, Alexa Fluor 488,Alexa Fluor 488, Alexa Fluor 488 hydrazide, Alexa Fluor 500, Alexa Fluor514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 555,Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 594,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 610-R-PE, Alexa Fluor 633,Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 647, Alexa Fluor 647-R-PE,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680-APC, Alexa Fluor680-R-PE, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790,Allophycocyanin, AmCyanl, Aminomethylcoumarin, Amplex Gold (product),Amplex Red Reagent, Amplex UltraRed, Anthracene, APC, APC-Seta-750,AsRed2, ATTO 390, ATTO 425, ATTO 430LS, ATTO 465, ATTO 488, ATTO 490LS,ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO 550, ATTO 565, ATTO 590,ATTO 594, ATTO 610, ATO 620, ATTO 633, ATTO 635, ATTO 647, ATTO 647N,ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, ATTO Oxa12,ATTO Rho3B, ATTO Rho6G, ATTO Rho11, ATTO Rho12, ATTO Rho13, ATTO Rho14,ATTO Rho101, ATTO Thio12, Auramine O, Azami Green, Azami Greenmonomeric, B-phycoerythrin, BCECF, BCECF, Bex1, Biphenyl, Birch Yellow580, Blue-green algae, BO-PRO-1, BO-PRO-3, BOBO-1, BOBO-3, BODIPY 630650-X, BODIPY 650/665-X, BODIPY FL, BODIPY FL, BODIPY R6G, BODIPY TMR-X,BODIPY TR-X, BODIPY TR-X Ph 7.0, BODIPY TR-X phallacidin, BODIPY-DiMe,BODIPY-Phenyl, BODIPY-TMSCC, C3-Indocyanine, C3-Indocyanine,C3-Oxacyanine, C3-Thiacyanine Dye (EtOH), C3-Thiacyanine Dye (PrOH),C5-Indocyanine, C5-Oxacyanine, C5-Thiacyanine, C7-Indocyanine,C7-Oxacyanine, C545T, C-Phycocyanin, Calcein, Calcein red-orange,Calcium Crimson, Calcium Green-1, Calcium Orange, Calcofluor white 2MR,Carboxy SNARF-1 pH 6.0, Carboxy SNARF-1 pH 9.0,Carboxynaphthofluorescein, Cascade Blue, Cascade Yellow, Catskill Green540, CBQCA, CellMask Orange, CellTrace BODIPY TR methyl ester, CellTracecalcein violet, CellTrace™ Far Red, CellTracker Blue, CellTracker RedCMTPX, CellTracker Violet BMQC, CF405M, CF405S, CF488A, CF543, CF555,CFP, CFSE, CF™ 350, CF™ 485, Chlorophyll A, Chlorophyll B, Chromeo 488,Chromeo 494, Chromeo 505, Chromeo 546, Chromeo 642, Citrine, Citrine,ClOH butoxy aza-BODIPY, ClOH C12 aza-BODIPY, CM-H2DCFDA, Coumarin 1,Coumarin 6, Coumarin 6, Coumarin 30, Coumarin 314, Coumarin 334,Coumarin 343, Coumarine 545T, Cresyl Violet Perchlorate, CryptoLightCF1, CryptoLight CF2, CryptoLight CF3, CryptoLight CF4, CryptoLight CF5,CryptoLight CF6, Crystal Violet, Cumarin153, Cy2, Cy3, Cy3, Cy3,5, Cy3B,Cy3B, Cy3Cy5 ET, Cy5, Cy5, Cy5.5, Cy7, Cyanine3 NHS ester, Cyanine5carboxylic acid, Cyanine5 NHS ester, Cyclotella meneghiniana Kützing,CypHer5, CypHer5 pH 9.15, CyQUANT GR, CyTrak Orange, Dabcyl SE, DAF-FM,DAMC (Weiss), dansyl cadaverine, Dansyl Glycine (Dioxane), DAPI, DAPI,DAPI, DAPI, DAPI (DMSO), DAPI (H2O), Dapoxyl (2-aminoethyl)sulfonamide,DCI, DCM, DCM, DCM (acetonitrile), DCM (MeOH), DDAO, Deep Purple,di-8-ANEPPS, DiA, Dichlorotris(1,10-phenanthroline) ruthenium(II).DiCIOH C12 aza-BODIPY, DiClOHbutoxy aza-BODIPY, DiD, DiI, DiIC18(3),DiO, DiR Diversa Cyan-FP, Diversa Green-FP, DM-NERF pH 4.0, DOCI,Doxorubicin, DPP pH-Probe 590-7.5. DPP pH-Probe 590-9.0, DPP pH-Probe590-11.0, DPP pH-Probe 590-11.0, Dragon Green, DRAQ5, DsRed, DsRed.DsRed, DsRed-Express. DsRed-Express2, DsRed-Express T1, dTomato,DY-350XL, DY-480, DY-480XL MegaStokes, DY-485, DY-485XL MegaStokes,DY-490, DY-490XL MegaStokes, DY-500, DY-500XL MegaStokes, DY-520,DY-520XL MegaStokes, DY-547, DY-549P1, DY-549P1, DY-554, DY-555, DY-557,DY-557, DY-590, DY-590, DY-615, DY-630, DY-631, DY-633, DY-635, DY-636,DY-647, DY-649P1, DY-649P1, DY-650, DY-651, DY-656, DY-673, DY-675,DY-676, DY-680, DY-681, DY-700, DY-701, DY-730, DY-731, DY-750, DY-751,DY-776, DY-782, Dye-28, Dye-33, Dye-45, Dye-304, Dye-1041, DyLight 488,DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680,E2-Crimson, E2-Orange, E2-Red/Green, EBFP, ECF, ECFP, ECL Plus, eGFP,ELF 97, Emerald, Envy Green, Eosin, Eosin Y, epicocconone, EqFP611,Erythrosin-5-isothiocyanate, Ethidium bromide, ethidium homodimer-1,Ethyl Eosin, Ethyl Eosin, Ethyl Nile Blue A,Ethyl-p-Dimethylaminobenzoate, Ethyl-p-Dimethylaminobenzoate, Eu2O3nanoparticles, Eu (Soini), Eu(tta)3DEADIT, EvaGreen, EVOblue-30, EYFP,FAD, FITC, FITC, FlAsH (Adams), Flash Red EX, FIAsH-CCPGCC,FIAsH-CCXXCC, Fluo-3, Fluo-4, Fluo-5F, Fluorescein, Fluorescein 0.1NaOH, Fluorescein-Dibase, fluoro-emerald, Fluorol 5G, FluoSpheres blue,FluoSpheres crimson, FluoSpheres dark red, FluoSpheres orange,FluoSpheres red, FluoSpheres yellow-green, FM4-64 in CTC, FM4-64 in SDS,FM 1-43, FM 4-64, Fort Orange 600, Fura Red, Fura Red Ca free, fura-2,Fura-2 Ca free, Gadodiamide, Gd-Dtpa-Bma, Gadodiamide, Gd-Dtpa-Bma,GelGreen™, GelRed™, H9-40, HcRed1, Hemo Red 720, HiLyte Fluor 488,HiLyte Fluor 555, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750,HiLyte Plus 555, HiLyte Plus 647, HiLyte Plus 750, HmGFP, Hoechst 33258,Hoechst 33342, Hoechst-33258, Hoechst-33258, Hops Yellow 560, HPTS,HPTS, HPTS, HPTS, HPTS, indo-1, Indo-1 Ca free, Ir(Cn)2(acac),Ir(Cs)2(acac), IR-775 chloride, IR-806, Ir-OEP-CO-Cl, IRDye® 650 Alkyne,IRDye® 650 Azide, IRDye® 650 Carboxylate, IRDye® 650 DBCO, IRDye® 650Maleimide, IRDye® 650 NHS Ester, IRDye®, 680LT Carboxylate, IRDye® 680LTMaleimide, IRDye® 680LT NHS Ester, IRDye® 680RD Alkyne, IRDye® 680RDAzide, IRDye® 680RD Carboxylate, IRDye® 680RD DBCO, IRDye® 680RDMaleimide, IRDye® 680RD NHS Ester, IRDye® 700 phosphoramidite, IRDye®700DX, IRDye® 700DX, IRDye® 700DX Carboxylate, IRDye® 700DX NHS Ester,IRDye® 750 Carboxylate, IRDye® 750 Maleimide, IRDye® 750 NHS Ester,IRDye® 800 phosphoramidite, IRDye® 800CW, IRDye® 800CW Alkyne, IRDye®800CW Azide, IRDye® 800CW Carboxylate, IRDye® 800CW DBCO, IRDye® 800CWMaleimide, IRDye® 800CW NHS Ester, IRDye® 800RS, IRDye® 800RSCarboxylate, IRDye® 800RS NHS Ester, IRDye® QC-1 Carboxylate, IRDye®QC-1 NHS Ester, Isochrysis galbana-Parke, JC-1, JC-1, JOJO-1, JonamacRed Evitag T2, Kaede Green, Kaede Red, kusabira orange, Lake Placid 490,LDS 751. Lissamine Rhodamine (Weiss), LOLO-1, lucifer yellow CH, LuciferYellow CH, lucifer yellow CH, Lucifer Yellow CH Dilitium salt, LumioGreen, Lumio Red, Lumogen F Orange, Lumogen Red F300, Lumogen Red F300,LysoSensor Blue DND-192, LysoSensor Green DND-153, LysoSensor GreenDND-153, LysoSensor Yellow/Blue DND-160 pH 3, LysoSensor YellowBlueDND-160, LysoTracker Blue DND-22, LysoTracker Blue DND-22, LysoTrackerGreen DND-26, LysoTracker Red DND-99, LysoTracker Yellow HCK-123, MacounRed Evitag T2, Macrolex Fluorescence Red G, Macrolex Fluorescence Yellow10GN, Macrolex Fluorescence Yellow 10GN, Magnesium Green, MagnesiumOctaethylporphyrin, Magnesium Orange, Magnesium Phthalocyanine,Magnesium Phthalocyanine, Magnesium Tetramesitylporphyrin, MagnesiumTetraphenylporphyrin, malachite green isothiocyanate, Maple Red-Orange620, Marina Blue, mBanana, mBBr, mCherry, Merocyanine 540, Methyl green,Methyl green, Methyl green, Methylene Blue, Methylene Blue, mHoneyDew,MitoTracker Deep Red 633, MitoTracker Green FM, MitoTracker OrangeCMTMRos, MitoTracker Red CMXRos, monobromobimane, Monochlorobimane,Monoraphidium, mOrange, mOrange2, mPlum, mRaspberry, mRFP, mRFP1,mRFP1.2 (Wang), mStrawberry (Shaner), mTangerine (Shaner),N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide),NADH, Naphthalene, Naphthalene, Naphthofluorescein, Naphthofluorescein,NBD-X, NeuroTrace 500525, Nilblau perchlorate, nile blue, Nile Blue,Nile Blue (EtOH), nile red, Nile Red, Nile Red, Nile red, Nileblue A,NIR1, NIR2, NIR3, NIR4, NIR820, Octaethylporphyrin, OH butoxyaza-BODIPY, OHC12 aza-BODIPY, Orange Fluorescent Protein, Oregon Green488, Oregon Green 488 DHPE, Oregon Green 514, Oxazin1, Oxazin 750,Oxazine 1, Oxazine 170, P4-3, P-Quaterphenyl, P-Terphenyl, PA-GFP(post-activation), PA-GFP (pre-activation), Pacific Orange.Palladium(II) meso-tetraphenyl-tetrabenzoporphyrin, PdOEPK, PdTFPP,PerCP-Cy5.5, Perylene, Perylene, Perylene bisimide pH-Probe 550-5.0,Perylene bisimide pH-Probe 550-5.5, Perylene bisimide pH-Probe 550-6.5,Perylene Green pH-Probe 720-5.5, Perylene Green Tag pH-Probe 720-6.0,Perylene Orange pH-Probe 550-2.0, Perylene Orange Tag 550, Perylene RedpH-Probe 600-5.5, Perylenediimid, Perylene Green pH-Probe 740-5.5,Phenol, Phenylalanine, pHrodo, succinimidyl ester, Phthalocyanine,PicoGreen dsDNA quantitation reagent, Pinacyanol-lodide, Piroxicam,Platinum(III) tetraphenyltetrabenzoporphyrin, Plum Purple, PO-PRO-1,PO-PRO-3, POPO-1, POPO-3, POPOP, Porphin, PPO, Proflavin,PromoFluor-350, PromoFluor-405, PromoFluor-415, PromoFluor-488,PromoFluor-488 Premium, PromoFluor-488LSS, PromoFluor-500LSS,PromoFluor-505, PromoFluor-510LSS, PromoFluor-514LSS, PromoFluor-520LSS,PromoFluor-532, PromoFluor-546, PromoFluor-555, PromoFluor-590,PromoFluor-610, PromoFluor-633, PromoFluor-647, PromoFluor-670,PromoFluor-680, PromoFluor-700, PromoFluor-750, PromoFluor-770,PromoFluor-780, PromoFluor-840, propidium iodide, Protoporphyrin IX,PTIR475/UF, PTIR545/UF, PtOEP, PtOEPK, PtTFPP, Pyrene, QD525, QD565,QD585, QD605, QD655, QD705, QD800, QD903, QD PbS 950, QDot 525, QDot545, QDot 565, Qdot 585, Qdot 605, Qdot 625, Qdot 655, Qdot 705, Qdot800, QpyMe2, QSY 7, QSY 7, QSY 9, QSY 21, QSY 35, quinine, QuinineSulfate, Quinine sulfate, R-phycoerythrin, R-phycoerythrin.ReAsH-CCPGCC, ReAsH-CCXXCC, Red Beads (Weiss), Redmond Red. Resorufin,resorufin, rhod-2, Rhodamin 700 perchlorate, rhodamine, Rhodamine 6G,Rhodamine 6G, Rhodamine 101, rhodamine 110, Rhodamine 123, rhodamine123, Rhodamine B, Rhodamine B, Rhodamine Green, Rhodamine pH-Probe585-7.0, Rhodamine pH-Probe 585-75, Rhodamine phalloidin, RhodamineRed-X, Rhodamine Red-X, Rhodamine Tag pH-Probe 585-7.0, Rhodol Green,Riboflavin, Rose Bengal, Sapphire, SBFI, SBFI Zero Na, Scenedesmus sp.,SensiLight PBXL-1, SensiLight PBXL-3, Seta 633-NHS, Seta-633-NHS,SeTau-380-NHS, SeTau-647-NHS, Snake-Eye Red 900, SNIR1, SNIR2, SNIR3,SNIR4, Sodium Green, Solophenyl flavine 7GFE 500, Spectrum Aqua,Spectrum Blue, Spectrum FRed, Spectrum Gold, Spectrum Green, SpectrumOrange, Spectrum Red, Squarylium dye III, Stains All, Stilben derivate,Stilbene, Styryl8 perchlorate, Sulfo-Cyanine3 carboxylic acid,Sulfo-Cyanine3 carboxylic acid, Sulfo-Cyanine3 NHS ester, Sulfo-Cyanine5carboxylic acid, Sulforhodamine 101, sulforhodamine 101, SulforhodamineB, Sulforhodamine G, Suncoast Yellow, SuperGlo BFP, SuperGlo GFP, SurfGreen EX, SYBR Gold nucleic acid gel stain, SYBR Green I, SYPRO Ruby,SYTO 9, SYTO 11, SYTO 13, SYTO 16, SYTO 17, SYTO 45, SYTO 59, SYTO 60,SYTO 61, SYTO 62, SYTO 82, SYTO RNASelect, SYTO RNASelect, SYTOX Blue,SYTOX Green, SYTOX Orange, SYTOX Red, T-Sapphire, Tb (Soini), tCO,tdTomato, Terrylen, Terrvlendiimid, testdye, Tetra-t-Butylazaporphine,Tetra-t-Butylnaphthalocyanine, Tetracen,Tetrakis(o-Aminophenyl)Porphyrin, Tetramesitylporphyrin,Tetramethylrhodamine, tetramethylrhodamine, Tetraphenylporphyrin,Tetraphenylporphyrin, Texas Red, Texas Red DHPE, Texas Red-X,ThiolTracker Violet, Thionin acetate, TMRE, TO-PRO-1, TO-PRO-3, Toluene,Topaz (Tsien1998), TOTO-1, TOTO-3, Tris(2,2-Bipyridyl)Ruthenium(II)chloride, Tris(4,4-diphenyl-2,2-bipyridine) ruthenium(II) chloride,Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) TMS, TRITC (Weiss),TRITC Dextran (Weiss), Tryptophan, Tyrosine, Vex1, Vybrant DyeCycleGreen stain, Vybrant DyeCycle Orange stain, Vybrant DyeCycle Violetstain, WEGFP (post-activation), WellRED D2, WellRED D3, WellRED D4,WtGFP, WtGFP (Tsien1998), X-rhod-1, Yakima Yellow, YFP, YO-PRO-1,YO-PRO-3, YOYO-1, YoYo-1, YoYo-1 dsDNA, YoYo-1 ssDNA, YOYO-3, ZincOctaethylporphyrin, Zinc Phthalocyanine, Zinc Tetramesitylporphyrin,Zinc Tetraphenylporphyrin, ZsGreen1, or ZsYellow1.

In embodiments, the detectable label is a fluorescent dye. Inembodiments, the detectable label is a fluorescent dye capable ofexchanging energy with another fluorescent dye (e.g., fluorescenceresonance energy transfer (FRET) chromophores).

In embodiments, the detectable moiety is a moiety of a derivative of oneof the detectable moieties described immediately above, wherein thederivative differs from one of the detectable moieties immediately aboveby a modification resulting from the conjugation of the detectablemoiety to a compound described herein.

The term “cyanine” or “cyanine moiety” as described herein refers to acompound containing two nitrogen groups separated by a polymethinechain. In embodiments, the cyanine moiety has 3 methine structures (i.e.cyanine 3 or Cy3). In embodiments, the cyanine moiety has 5 methinestructures (i.e. cyanine 5 or Cy5). In embodiments, the cyanine moietyhas 7 methine structures (i.e. cyanine 7 or Cy7).

Descriptions of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included are salts of amino acids such as arginate and thelike, and salts of organic acids like glucuronic or galactunoric acidsand the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Non-limiting examples of such salts include hydrochlorides,hydrobromides, phosphates, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, propionates, tartrates (e.g.,(+)-tartrates, (−)-tartrates, or mixtures thereof including racemicmixtures), succinates, benzoates, and salts with amino acids such asglutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyliodide, and the like). These salts may be prepared by methods known tothose skilled in the art.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compound maydiffer from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Prodrugs of the compounds described herein may be convertedin vivo after administration. Additionally, prodrugs can be converted tothe compounds of the present invention by chemical or biochemicalmethods in an ex vivo environment, such as, for example, when contactedwith a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may optionally be conjugated to a moiety that doesnot consist of amino acids. The terms apply to amino acid polymers inwhich one or more amino acid residue is an artificial chemical mimeticof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymer.

A polypeptide, or a cell is “recombinant” when it is artificial orengineered, or derived from or contains an artificial or engineeredprotein or nucleic acid (e.g. non-natural or not wild type). Forexample, a polynucleotide that is inserted into a vector or any otherheterologous location, e.g., in a genome of a recombinant organism, suchthat it is not associated with nucleotide sequences that normally flankthe polynucleotide as it is found in nature is a recombinantpolynucleotide. A protein expressed in vitro or in vivo from arecombinant polynucleotide is an example of a recombinant polypeptide.Likewise, a polynucleotide sequence that does not appear in nature, forexample a variant of a naturally occurring gene, is recombinant.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents that can be producedin the reaction mixture. The term “contacting” may include allowing twospecies to react, interact, or physically touch, wherein the two speciesmay be a compound as described herein and a protein or enzyme. In someembodiments contacting includes allowing a compound described herein tointeract with a protein or enzyme that is involved in a signalingpathway.

As defined herein, the term “activation”, “activate”, “activating” andthe like in reference to a protein refers to conversion of a proteininto a biologically active derivative from an initial inactive ordeactivated state. The terms reference activation, or activating,sensitizing, or up-regulating signal transduction or enzymatic activityor the amount of a protein decreased in a disease.

The terms “agonist,” “activator,” “upregulator,” etc. refer to asubstance capable of detectably increasing the expression or activity ofa given gene or protein. The agonist can increase expression or activity10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to acontrol in the absence of the agonist. In certain instances, expressionor activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold orhigher than the expression or activity in the absence of the agonist.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor interaction meansnegatively affecting (e.g. decreasing) the activity or function of theprotein relative to the activity or function of the protein in theabsence of the inhibitor. In embodiments inhibition means negativelyaffecting (e.g. decreasing) the concentration or levels of the proteinrelative to the concentration or level of the protein in the absence ofthe inhibitor. In embodiments inhibition refers to reduction of adisease or symptoms of disease. In embodiments, inhibition refers to areduction in the activity of a particular protein target. Thus,inhibition includes, at least in part, partially or totally blockingstimulation, decreasing, preventing, or delaying activation, orinactivating, desensitizing, or down-regulating signal transduction orenzymatic activity or the amount of a protein. In embodiments,inhibition refers to a reduction of activity of a target proteinresulting from a direct interaction (e.g. an inhibitor binds to thetarget protein). In embodiments, inhibition refers to a reduction ofactivity of a target protein from an indirect interaction (e.g. aninhibitor binds to a protein that activates the target protein, therebypreventing target protein activation).

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator”interchangeably refer to a substance capable of detectably decreasingthe expression or activity of a given gene or protein. The antagonistcan decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more in comparison to a control in the absence of theantagonist. In certain instances, expression or activity is 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression oractivity in the absence of the antagonist.

The terms “streptavidin” and “

” refer to a tetrameric protein (including homologs, isoforms, andfunctional fragments thereof) capable of binding biotin. The termincludes any recombinant or naturally-occurring form of streptavidinvariants thereof that maintain streptavidin activity (e.g. within atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity comparedto wildtype streptavidin).

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion. Expression can be detected usingconventional techniques for detecting protein (e.g. ELISA, Westernblotting, flow cytometry, immunofluorescence, immunohistochemistry,etc.).

An “effective amount” is an amount sufficient for a compound toaccomplish a stated purpose relative to the absence of the compound(e.g. achieve the effect for which it is administered, treat a disease,reduce enzyme activity, increase enzyme activity, reduce a signalingpathway, or reduce one or more symptoms of a disease or condition). An“activity decreasing amount,” as used herein, refers to an amount ofantagonist required to decrease the activity of an enzyme relative tothe absence of the antagonist. A “function disrupting amount.” as usedherein, refers to the amount of antagonist required to disrupt thefunction of an enzyme or protein relative to the absence of theantagonist.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaroytic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells. Cells maybe useful when they are naturally nonadherent or have been treated notto adhere to surfaces, for example by trypsinization.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects. In some embodiments, acontrol is the measurement of the activity of a protein in the absenceof a compound as described herein (including embodiments and examples).

The term “modulate” is used in accordance with its plain ordinarymeaning and refers to the act of changing or varying one or moreproperties. “Modulation” refers to the process of changing or varyingone or more properties. For example, as applied to the effects of amodulator on a target protein, to modulate means to change by increasingor decreasing a property or function of the target molecule or theamount of the target molecule.

The term “aberrant” as used herein refers to different from normal. Whenused to describe enzymatic activity or protein function, aberrant refersto activity or function that is greater or less than a normal control orthe average of normal non-diseased control samples.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents used herein means at least two nucleotides covalently linkedtogether. The term “nucleic acid” includes single-, double-, ormultiple-stranded DNA, RNA and analogs (derivatives) thereof.Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25,30, 40, 50 or more nucleotides in length, up to about 100 nucleotides inlength. Nucleic acids and polynucleotides are a polymers of any length,including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000,7000, 10,000, etc. In certain embodiments the nucleic acids hereincontain phosphodiester bonds. In other embodiments, nucleic acid analogsare included that may have alternate backbones, comprising, e.g.,phosphoramidate, phosphorothioate, phosphorodithioate, orO-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press); and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g. to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. A residue of a nucleic acid, as referred to herein,is a monomer of the nucleic acid (e.g., a nucleotide).

A particular nucleic acid sequence also encompasses “splice variants.”Similarly, a particular protein encoded by a nucleic acid encompassesany protein encoded by a splice variant of that nucleic acid. “Splicevariants,” as the name suggests, are products of alternative splicing ofa gene. After transcription, an initial nucleic acid transcript may bespliced such that different (alternate) nucleic acid splice productsencode different polypeptides. Mechanisms for the production of splicevariants vary, but include alternate splicing of exons. Alternatepolypeptides derived from the same nucleic acid by read-throughtranscription are also encompassed by this definition. Any products of asplicing reaction, including recombinant forms of the splice products,are included in this definition. An example of potassium channel splicevariants is discussed in Leicher, et al., J. Biol. Chem.273(52):35095-35101 (1998).

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are near each other, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or higher identity over a specified region whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see. e.g., NCBI web site or thelike). Such sequences are then said to be “substantially identical.”This definition also refers to, or may be applied to, the compliment ofa test sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about10 amino acids or 20 nucleotides in length, or more preferably over aregion that is 10-50 amino acids or 20-50 nucleotides in length. As usedherein, percent (%) amino acid sequence identity is defined as thepercentage of amino acids in a candidate sequence that are identical tothe amino acids in a reference sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)software. Appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared can be determined by known methods.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 10 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch. J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr, Madison,Wis.), or by manual alignment and visual inspection (see. e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds, 1995 supplement)).

As used herein, the term “bioconjugate” or “bioconjugate linker” refersto the resulting association between atoms or molecules of bioconjugatereactive groups. The association can be direct or indirect. For example,a conjugate between a first bioconjugate reactive group (e.g. —NH₂,—COOH, —N-hydroxysuccinimide, or -maleimide) and a second bioconjugatereactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine,amine sidechain containing amino acid, or carboxylate) provided hereincan be direct, e.g., by covalent bond or linker (e.g. a first linker ofsecond linker), or indirect, e.g., by non-covalent bond (e.g.electrostatic interactions (e.g. ionic bond, hydrogen bond, halogenbond), van der Waals interactions (e.g. dipole-dipole, dipole-induceddipole, London dispersion), ring stacking (pi effects), hydrophobicinteractions and the like). In embodiments a bioconjugate is a clickchemistry reactant moiety when the association between atoms ormolecules of bioconjugate reactive groups is direct (e.g., covalentbond, linker).

In embodiments, bioconjugates or bioconjugate linkers are formed usingbioconjugate chemistry (i.e. the association of two bioconjugatereactive groups) including, but are not limited to nucleophilicsubstitutions (e.g., reactions of amines and alcohols with acyl halides,active esters), electrophilic substitutions (e.g., enamine reactions)and additions to carbon-carbon and carbon-heteroatom multiple bonds(e.g., Michael reaction, Diels-Alder addition). These and other usefulreactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington. D.C., 1982. In embodiments, thefirst bioconjugate reactive group (e.g., maleimide moiety) is covalentlyattached to the second bioconjugate reactive group (e.g. a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g., haloacetylmoiety) is covalently attached to the second bioconjugate reactive group(e.g. a sulfhydryl). In embodiments, the first bioconjugate reactivegroup (e.g., pyridyl moiety) is covalently attached to the secondbioconjugate reactive group (e.g. a sulfhydryl). In embodiments, thefirst bioconjugate reactive group (e.g., —N-hydroxysuccinimide moiety)is covalently attached to the second bioconjugate reactive group (e.g.an amine). In embodiments, the first bioconjugate reactive group (e.g.,maleimide moiety) is covalently attached to the second bioconjugatereactive group (e.g. a sulfhydryl). In embodiments, the firstbioconjugate reactive group (e.g., -sulfo-N-hydroxysuccinimide moiety)is covalently attached to the second bioconjugate reactive group (e.g.an amine).

Useful bioconjugate reactive groups used for bioconjugate chemistriesherein include, for example: (a) carboxyl groups and various derivativesthereof including, but not limited to, N-hydroxysuccinimide esters,N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters,p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b)hydroxyl groups which can be converted to esters, ethers, aldehydes,etc; (c) haloalkyl groups wherein the halide can be later displaced witha nucleophilic group such as, for example, an amine, a carboxylateanion, thiol anion, carbanion, or an alkoxide ion, thereby resulting inthe covalent attachment of a new group at the site of the halogen atom;(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido or maleimide groups; (e)aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition; (f) sulfonyl halide groupsfor subsequent reaction with amines, for example, to form sulfonamides;(g) thiol groups, which can be converted to disulfides, reacted withacyl halides, or bonded to metals such as gold, or react withmaleimides; (h) amine or sulfhydryl groups (e.g., present in cysteine),which can be, for example, acylated, alkylated or oxidized; (i) alkenes,which can undergo, for example, cycloadditions, acylation, Michaeladdition, etc; (j) epoxides, which can react with, for example, aminesand hydroxyl compounds; (k) phosphoramidites and other standardfunctional groups useful in nucleic acid synthesis; (1) metal siliconoxide bonding; (m) metal bonding to reactive phosphorus groups (e.g.phosphines) to form, for example, phosphate diester bonds.; (n) azidescoupled to alkynes using copper catalyzed cycloaddition click chemistry;(o) biotin conjugate can react with avidin or strepavidin to form aavidin-biotin complex or streptavidin-biotin complex.

The bioconjugate reactive groups can be chosen such that they do notparticipate in, or interfere with, the chemical stability of theconjugate described herein. Alternatively, a reactive functional groupcan be protected from participating in the crosslinking reaction by thepresence of a protecting group. In embodiments, the bioconjugatecomprises a molecular entity derived from the reaction of an unsaturatedbond, such as a maleimide, and a sulfhydryl group.

The terms “monophosphate” is used in accordance with its ordinarymeaning in the arts and refers to a moiety having the formula:

The term “polyphosphate” refers to at least two phosphate groups, havingthe formula:

wherein np is an integer of 1 or greater. In embodiments, np is aninteger from 0 to 5. In embodiments, np is an integer from 0 to 2. Inembodiments, np is 2.

The term “base” as used herein refers to a divalent purine or pyrimidinecompound or a derivative thereof, that may be a constituent of nucleicacid (i.e. DNA or RNA, or a derivative thereof). In embodiments, thebase is a derivative of a naturally occurring DNA or RNA base (e.g., abase analogue). In embodiments the base is a hybridizing base. Inembodiments the base hybridizes to a complementary base. In embodiments,the base is capable of forming at least one hydrogen bond with acomplementary base (e.g., adenine hydrogen bonds with thymine, adeninehydrogen bonds with uracil, guanine pairs with cytosine). Non-limitingexamples of a base includes cytosine or a derivative thereof (e.g.,cytosine analogue), guanine or a derivative thereof (e.g., guanineanalogue), adenine or a derivative thereof (e.g., adenine analogue),thymine or a derivative thereof (e.g., thymine analogue), uracil or aderivative thereof (e.g., uracil analogue), hypoxanthine or a derivativethereof (e.g., hypoxanthine analogue), xanthine or a derivative thereof(e.g., xanthine analogue), 7-methylguanine or a derivative thereof(e.g., 7-methylguanine analogue), deaza-adenine or a derivative thereof(e.g., deaza-adenine analogue), deaza-guanine or a derivative thereof(e.g., deaza-guanine), deaza-hypoxanthine or a derivative thereof,5,6-dihydrouracil or a derivative thereof (e.g., 5,6-dihydrouracilanalogue), 5-methylcytosine or a derivative thereof (e.g.,5-methylcytosine analogue), or 5-hydroxymethylcytosine or a derivativethereof (e.g., 5-hydroxymethylcytosine analogue) moieties. Inembodiments, the base is adenine, guanine, hypoxanthine, xanthine,theobromine, caffeine, uric acid, or isoguanine. In embodiments, thebase is

The term “non-covalent linker” is used in accordance with its ordinarymeaning and refers to a divalent moiety which includes at least twomolecules that are not covalently linked to each other but do interactwith each other via a non-covalent bond (e.g. electrostatic interactions(e.g. ionic bond, hydrogen bond, halogen bond) or van der Waalsinteractions (e.g. dipole-dipole, dipole-induced dipole, Londondispersion).

The term “anchor moiety” as used herein refers to a chemical moietycapable of interacting (e.g., covalently or non-covalently) with asecond, optionally different, chemical moiety (e.g., complementaryanchor moiety binder). In embodiments, the anchor moiety is abioconjugate reactive group capable of interacting (e.g., covalently)with a complementary bioconjugate reactive group (e.g., complementaryanchor moiety reactive group). In embodiments, an anchor moiety is aclick chemistry reactant moiety. In embodiments, the anchor moiety (an“affinity anchor moiety”) is capable of non-covalently interacting witha second chemical moiety (e.g., complementary affinity anchor moietybinder). Non-limiting examples of an anchor moiety include biotin,azide, trans-cyclooctene (TCO) and phenyl boric acid (PBA). Inembodiments, an affinity anchor moiety (e.g., biotin moiety) interactsnon-covalently with a complementary affinity anchor moiety binder (e.g.,streptavidin moiety). In embodiments, an anchor moiety (e.g., azidemoiety, trans-cyclooctene (TCO) moiety, phenyl boric acid (PBA) moiety)covalently binds a complementary anchor moiety binder (e.g.,dibenzocyclooctyne (DBCO) moiety, tetrazine (TZ) moiety,salicylhydroxamic acid (SHA) moiety).

The terms “cleavable linker” or “cleavable moiety” as used herein refersto a divalent or monovalent, respectively, moiety which is capable ofbeing separated (e.g., detached, split, disconnected, hydrolyzed, astable bond within the moiety is broken) into distinct entities. Acleavable linker is cleavable (e.g., specifically cleavable) in responseto external stimuli (e.g., enzymes, nucleophilic/basic reagents,reducing agents, photo-irradiation, electrophilic/acidic reagents,organometallic and metal reagents, or oxidizing reagents). A chemicallycleavable linker refers to a linker which is capable of being split inresponse to the presence of a chemical (e.g., acid, base, oxidizingagent, reducing agent, Pd(0), tris-(2-carboxyethyl)phosphine, dilutenitrous acid, fluoride, tris(3-hydroxypropyl)phosphine), sodiumdithionite (Na₂S₂O₄), hydrazine (N₂H₄)). A chemically cleavable linkeris non-enzymatically cleavable. In embodiments, the cleavable linker iscleaved by contacting the cleavable linker with a cleaving agent. Inembodiments, the cleaving agent is sodium dithionite (Na₂S₂O₄), weakacid, hydrazine (N₂H₄), Pd(0), or light-irradiation (e.g., ultravioletradiation).

A photocleavable linker (e.g., including or consisting of ao-nitrobenzyl group) refers to a linker which is capable of being splitin response to photo-irradiation (e.g., ultraviolet radiation). Anacid-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., increased acidity). Abase-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., decreased acidity). Anoxidant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of an oxidizing agent. Areductant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of an reducing agent (e.g.,Tris(3-hydroxypropyl)phosphine). In embodiments, the cleavable linker isa dialkylketal linker, an azo linker, an allyl linker, a cyanoethyllinker, a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

The term “orthogonally cleavable linker” or “orthogonal cleavablelinker” as used herein refer to a cleavable linker that is cleaved by afirst cleaving agent (e.g., enzyme, nucleophilic/basic reagent, reducingagent, photo-irradiation, electrophilic/acidic reagent, organometallicand metal reagent, oxidizing reagent) in a mixture of two or moredifferent cleaving agents and is not cleaved by any other differentcleaving agent in the mixture of two or more cleaving agents. Forexample, two different cleavable linkers are both orthogonal cleavablelinkers when a mixture of the two different cleavable linkers arereacted with two different cleaving agents and each cleavable linker iscleaved by only one of the cleaving agents and not the other cleavingagent. In embodiments, an orthogonally is a cleavable linker thatfollowing cleavage the two separated entities (e.g., fluorescent dye,bioconjugate reactive group) do not further react and form a neworthogonally cleavable linker.

The term “orthogonal binding group” or “orthogonal binding molecule” asused herein refer to a binding group (e.g. anchor moiety orcomplementary anchor moiety binder) that is capable of binding a firstcomplementary binding group (e.g., complementary anchor moiety binder oranchor moiety) in a mixture of two or more different complementarybinding groups and is unable to bind any other different complementarybinding group in the mixture of two or more complementary bindinggroups. For example, two different binding groups are both orthogonalbinding groups when a mixture of the two different binding groups arereacted with two complementary binding groups and each binding groupbinds only one of the complementary binding groups and not the othercomplementary binding group. An example of a set of four orthogonalbinding groups and a set of orthogonal complementary binding groups arethe binding groups biotin, azide, trans-cyclooctene (TCO) and phenylboric acid (PBA), which specifically and efficiently bind or react withthe complementary binding groups streptavidin, dibenzocyclooctyne(DBCO), tetrazine (TZ) and salicylhydroxamic acid (SHA) respectively.

The term “orthogonal detectable label” or “orthogonal detectable moiety”as used herein refer to a detectable label (e.g. fluorescent dye ordetectable dye) that is capable of being detected and identified (e.g.,by use of a detection means (e.g., emission wavelength, physicalcharacteristic measurement)) in a mixture or a panel (collection ofseparate samples) of two or more different detectable labels. Forexample, two different detectable labels that are fluorescent dyes areboth orthogonal detectable labels when a panel of the two differentfluorescent dyes is subjected to a wavelength of light that is absorbedby one fluorescent dye but not the other and results in emission oflight from the fluorescent dye that absorbed the light but not the otherfluorescent dye. Orthogonal detectable labels may be separatelyidentified by different absorbance or emission intensities of theorthogonal detectable labels compared to each other and not only be theabsolute presence of absence of a signal. An example of a set of fourorthogonal detectable labels is the set of Rox-Labeled Tetrazine,Alexa488-Labeled SHA. Cy5-Labeled Streptavidin, and R6G-LabeledDibenzocyclooctyne.

The term “polymerase-compatible cleavable moiety” as used herein refersa cleavable moiety which does not interfere with the function of apolymerase (e.g., DNA polymerase, modified DNA polymerase). Methods fordetermining the function of a polymerase contemplated herein aredescribed in B. Rosenblum et al. (Nucleic Acids Res. 1997 Nov. 15;25(22): 4500-4504); and Z. Zhu et al. (Nucleic Acids Res. 1994 Aug. 25;22(16): 3418-3422), which are incorporated by reference herein in theirentirety for all purposes. In embodiments the polymerase-compatiblecleavable moiety does not decrease the function of a polymerase relativeto the absence of the polymerase-compatible cleavable moiety. Inembodiments, the polymerase-compatible cleavable moiety does notnegatively affect DNA polymerase recognition. In embodiments, thepolymerase-compatible cleavable moiety does not negatively affect (e.g.,limit) the read length of the DNA polymerase. Additional examples of apolymerase-compatible cleavable moiety may be found in U.S. Pat. No.6,664,079, Ju J. et al. (2006) Proc Natl Acad Sci USA103(52):19635-19640.; Ruparel H. et al. (2005) Proc Natl Acad Sci USA102(17):5932-5937.; Wu J. et al. (2007) Proc Natl Acad Sci USA 104(104):16462-16467; Guo J. et al. (2008) Proc Natl Acad Sci USA 105(27):9145-9150 Bentley D. R. et al. (2008) Nature 456(7218):53-59; or HutterD. et al. (2010) Nucleosides Nucleotides & Nucleic Acids 29:879-895,which are incorporated herein by reference in their entirety for allpurposes. In embodiments, a polymerase-compatible cleavable moietyincludes an azido moiety or a dithiol linking moiety. In embodiments,the polymerase-compatible cleavable moiety is —NH₂, —CN, —CH₃, C₂-C₆allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g., —CH₂—O—CH₃), or —CH₂N₃.In embodiments, the polymerase-compatible cleavable moiety is:

The term “allyl” as described herein refers to an unsubstitutedmethylene attached to a vinyl group (i.e. —CH═CH₂), having the formula

An “allyl linker” refers to a divalent unsubstituted methylene attachedto a vinyl group, having the formula

The term “polymerase-compatible moiety” as used herein refers a moietywhich does not interfere with the function of a polymerase (e.g., DNApolymerase, modified DNA polymerase). Methods for determining thefunction of a polymerase contemplated herein are described in B.Rosenblum et al. (Nucleic Acids Res. 1997 Nov. 15; 25(22): 4500-4504);and Z. Zhu et al. (Nucleic Acids Res. 1994 Aug. 25; 22(16): 3418-3422),which are incorporated by reference herein in their entirety for allpurposes. In embodiments the polymerase-compatible moiety does notdecrease the function of a polymerase relative to the absence of thepolymerase-compatible moiety. In embodiments, the polymerase-compatiblemoiety does not negatively affect DNA polymerase recognition. Inembodiments, the polymerase-compatible moiety does not negatively affect(e.g., limit) the read length of the DNA polymerase. Additional examplesof a polymerase-compatible moiety may be found in U.S. Pat. No.6,664,079, Ju J. et al. (2006) Proc Natl Acad Sci USA103(52):19635-19640.; Ruparel H. et al. (2005) Proc Natl Acad Sci USA102(17):5932-5937.; Wu J. et al. (2007) Proc Natl Acad Sci USA104(104):16462-16467; Guo J. et al. (2008) Proc Natl Acad Sci USA105(27): 9145-9150 Bentley D. R. et al. (2008) Nature 456(7218):53-59;or Hutter D. et al. (2010) Nucleosides Nucleotides & Nucleic Acids29:879-895, which are incorporated herein by reference in their entiretyfor all purposes.

The term “thermophilic nucleic acid polymerase” as used herein refers toa family of DNA polymerases (e.g., 9° N™) and mutants thereof derivedfrom the DNA polymerase originally isolated from the hyperthermophilicarchaea, Thermococcus sp. 9 degrees N-7, found in hydrothermal vents atthat latitude (East Pacific Rise) (Southworth M W, et al. PNAS. 1996;93(11):5281-5285). A thermophilic nucleic acid polymerase is a member ofthe family B DNA polymerases. Site-directed mutagenesis of the 3′-5′ exomotif I (Asp-Ile-Glu) to Asp-Ile-Asp resulted in reduction of 3′-5′exonuclease activity to <1% of wild-type, while maintaining otherproperties of the polymerase including its high strand displacementactivity. Subsequent mutagenesis of key amino acids results in anincreased ability of the enzyme to incorporate dideoxynucleotides,ribonucleotides and acyclonucleotides (e.g., Therminator II enzyme fromNew England Biolabs with D141A/E143A/Y409V/A485L mutations);3′-amino-dNTPs, 3′-azido-dNTPs and other 3′-modified nucleotides (e.g.,NEB Therminator III DNA Polymerase with D141A/E143A/L408S/Y409A/P410Vmutations. NEB Therminator IX DNA polymerase), or γ-phosphate labelednucleotides (e.g., Therminator γ:D141A/E143A/W355A/L408W/R460A/Q461S/K464E/D480V/R484W/A485L). Typicallythese enzymes do not have 5′-3′ exonuclease activity. Additionalinformation about thermophilic nucleic acid polymerases may be found in(Southworth M W, et al. PNAS. 1996; 93(11):5281-5285; Bergen K, et al.Chem Bio Chem. 2013; 14(9):1058-1062; Kumar S, et al. ScientificReports. 2012; 2:684; Fuller C W, et al. 2016; 113(19):5233-5238; Guo J,et al. Proceedings of the National Academy of Sciences of the UnitedStates of America. 2008:105(27):9145-9150), which are incorporatedherein in their entirety for all purposes.

The term “primer”, as used herein, is defined to be one or more nucleicacid fragments that specifically hybridize to a nucleic acid template. Aprimer can be of any length depending on the particular technique itwill be used for. For example, PCR primers are generally between 10 and40 nucleotides in length. The length and complexity of the nucleic acidfixed onto the nucleic acid template is not critical to the invention.One of skill can adjust these factors to provide optimum hybridizationand signal production for a given hybridization procedure, and toprovide the required resolution among different genes or genomiclocations.

The phrase “stringent hybridization conditions” refers to conditionsunder which a primer will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, ed. Ausubel, et al., supra.

II. Compositions

In an aspect is provided a nucleotide analogue having the formula:

B is a base or analogue thereof. L¹ is covalent linker. L² is covalentlinker. L⁴ is covalent linker. X is a bond, O, NR^(6A), or S. R³ is —OH,monophosphate, polyphosphate or a nucleic acid. R^(4A) and R^(6A) areindependently hydrogen, —OH, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂,—CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁵ is a detectable label, anchor moiety, oraffinity anchor moiety. R⁶ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁷ is hydrogen or —OR^(7A), wherein R^(7A) ishydrogen or a polymerase-compatible moiety. R¹² is a complementaryaffinity anchor moiety binder. R¹³ is a detectable label. The symbol“----” is a non-covalent bond.

In embodiments, the nucleotide analogue has the formula:

wherein R³, B, R⁷, L¹, R^(4A), X, R⁶, L², and R⁵ are as describedherein. In embodiments, R⁵ is a detectable label or anchor moiety.

In embodiments, the nucleotide analogue has the formula:

wherein R³, B, R⁷, L¹, R^(4A), X, R⁶, L², R⁵, R¹², L⁴, and R¹³ are asdescribed herein. In embodiments, R⁵ is an affinity anchor moiety. Thesymbol “----” is a non-covalent bond.

In an aspect is provided a nucleotide analogue having the formula:

wherein L³ is a cleavable linker; R³ is —OH, monophosphate,polyphosphate or a nucleic acid; B is a base or analogue thereof; R⁵ isa detectable label or anchor moiety; and R⁷ is hydrogen or —OR^(7A),wherein R^(7A) is hydrogen or a polymerase-compatible moiety.

In an aspect is provided a nucleotide analogue having the formula:

wherein L³ is a cleavable linker; R³ is —OH, monophosphate,polyphosphate or a nucleic acid; B is a base or analogue thereof; R⁵ isa detectable label or anchor moiety and R⁷ is hydrogen or —OR^(7A),wherein R^(7A) is hydrogen or a polymerase-compatible moiety. L⁴ iscovalent linker. R¹² is a complementary affinity anchor moiety binder.R¹³ is a detectable label. The symbol “----” is a non-covalent bond.

In an aspect is provided a nucleic acid polymerase comprisingnon-thermophilic or thermophilic polymerase that forms a ternary complexwith the primed template and the nucleotide analogue, wherein thenucleic acid polymerase is bound to a nucleotide analogue having theformula:

wherein R³, B, R⁷, L¹, R^(4A), X, R⁶, L², and R⁵ are as describedherein, or

wherein R³, B, R⁷, L¹, R^(4A), R⁵, L², R⁵, R¹², L⁴, and R¹³ are asdescribed herein.

In embodiments, the nucleotide analogue has the formula:

wherein R³, B, R⁷, L¹, R^(4A), R⁶, L², and R⁵ are as described herein.In embodiments, R⁵ is a detectable label or anchor moiety. Inembodiments, R^(4A) is not hydrogen. In embodiments, R^(4B) is nothydrogen. In embodiments, R^(4A) and R^(4B) are not hydrogen.

In embodiments, the nucleotide analogue has the formula:

wherein R³, B, R⁷, L¹, R^(4A), R⁶, L², R⁵, R¹², L⁴, and R¹³ are asdescribed herein. In embodiments, R⁵ is an affinity anchor moiety. Thesymbol “----” is a non-covalent bond. In embodiments, R^(4A) is nothydrogen. In embodiments, R⁶ is not hydrogen. In embodiments, R^(4A) andR⁶ are not hydrogen.

In an aspect is provided a nucleic acid polymerase (e.g., thermophilic,9° N and mutants thereof, Phi29 and mutants thereof) complex, whereinthe thermophilic nucleic acid polymerase is bound to a nucleotideanalogue having the formula:

B is a base or analogue thereof. L¹ is covalent linker. L² is covalentlinker. L⁴ is covalent linker. R³ is —OH, monophosphate, polyphosphateor a nucleic acid. R^(4A) and R^(6A) are independently is hydrogen, —OH,—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl,—CH₂Br, —CH₂I, —CN, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R^(4B)is hydrogen, —OH, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂,—CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —X—R⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. X is a bond, O, NR^(6A), or S. R⁵ is adetectable label, anchor moiety, or affinity anchor moiety. R⁶ ishydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F,—CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁷ ishydrogen or —OR^(7A), wherein R^(7A) is hydrogen or apolymerase-compatible moiety. R¹² is a complementary affinity anchormoiety binder. R¹³ is a detectable label. The symbol “----” is anon-covalent bond.

In an aspect is provided a thermophilic nucleic acid polymerase complex,wherein the thermophilic nucleic acid polymerase is bound to anucleotide analogue having the formula:

B is a base or analogue thereof. L¹ is covalent linker. L² is covalentlinker. L⁴ is covalent linker. R³ is —OH, monophosphate, polyphosphateor a nucleic acid. R^(4A) and R^(6A) are independently is hydrogen, —OH,—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl,—CH₂Br, —CH₂I, —CN, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R^(4B)is hydrogen, —OH, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂,—CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —X—R⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. X is a bond, O, NR^(6A), or S. R⁵ is adetectable label, anchor moiety, or affinity anchor moiety. R⁶ ishydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F,—CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁷ ishydrogen or —OR^(1A), wherein R^(7A) is hydrogen or apolymerase-compatible moiety. R¹² is a complementary affinity anchormoiety binder. R¹³ is a detectable label. The symbol “----” is anon-covalent bond.

In embodiments, the thermophilic nucleic acid polymerase is bound to anucleotide analogue having the formula:

wherein R³, B, R⁷, L¹, R^(4A), R^(4B), L², and R⁵ are as describedherein. In embodiments, R⁵ is a detectable label or anchor moiety.

In embodiments, the thermophilic nucleic acid polymerase is bound to anucleotide analogue having the formula:

wherein R³, B, R⁷, L¹, R^(4A), R^(4B), L², R⁵, R¹², L⁴, and R¹³ are asdescribed herein. In embodiments, R⁵ is an affinity anchor moiety. Thesymbol “----” is a non-covalent bond.

In another aspect is provided a thermophilic nucleic acid polymerasecomplex (e.g., 9°N nucleic acid polymerase complex), wherein thethermophilic nucleic acid polymerase is bound to a nucleotide analogue,wherein the nucleotide analogue includes a fluorescent dye with amolecular weight of at least about 140 Daltons, and wherein thefluorescent dye is covalently bound at the 3′ position of the nucleotideanalogue. In embodiments, the fluorescent dye is covalently bound at the3′ position of the nucleotide analogue via a linker (e.g., —S(O)₂—,—NH—, —O—, —S—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —NHC(O)NH—,—C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene).

In embodiments, B is cytosine or a derivative thereof, guanine or aderivative thereof, adenine or a derivative thereof, thymine or aderivative thereof, uracil or a derivative thereof, hypoxanthine or aderivative thereof, xanthine or a derivative thereof, deaza-adenine or aderivative thereof, deaza-guanine or a derivative thereof,deaza-hypoxanthine or a derivative thereof, 7-methylguanine or aderivative thereof, 5,6-dihydrouracil or a derivative thereof,5-methylcytosine or a derivative thereof, or 5-hydroxymethylcytosine ora derivative thereof.

In embodiments, B is cytosine or a derivative thereof. In embodiments, Bis guanine or a derivative thereof. In embodiments, B is adenine or aderivative thereof. In embodiments. B is thymine or a derivativethereof. In embodiments, B is uracil or a derivative thereof. Inembodiments, B is hypoxanthine or a derivative thereof. In embodiments,B is xanthine or a derivative thereof. In embodiments, B isdeaza-adenine or a derivative thereof. In embodiments, B isdeaza-guanine or a derivative thereof. In embodiments, B isdeaza-hypoxanthine or a derivative thereof. In embodiments, B is7-methylguanine or a derivative thereof. In embodiments, B is5,6-dihydrouracil or a derivative thereof. In embodiments, B is5-methylcytosine or a derivative thereof. In embodiments, B is or5-hydroxymethylcytosine or a derivative thereof.

In embodiments, B is cytosine, guanine, adenine, thymine, uracil,hypoxanthine, xanthine, deaza-adenine, deaza-guanine, deaza-hypoxanthineor a derivative thereof, 7-methylguanine, 5,6-dihydrouracil,5-methylcytosine, or 5-hydroxymethylcytosine. In embodiments, B iscytosine. In embodiments, B is guanine. In embodiments, B is adenine. Inembodiments, B is thymine. In embodiments, B is uracil. In embodiments,B is hypoxanthine. In embodiment, B is xanthine. In embodiments, B isdeaza-adenine. In embodiments, B is deaza-guanine. In embodiments, B isdeaza-hypoxanthine. In embodiments, B is 7-methylguanine. Inembodiments, B is 5,6-dihydrouracil. In embodiments, B is5-methylcytosine. In embodiments, B is or 5-hydroxymethylcytosine.

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substitutedor unsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; wherein at least one ofL^(1A), L^(1B), L^(1C), L^(1D) and L^(1E) is not a bond.

In embodiments, L¹ is a substituted or unsubstituted methylene, whereinL¹ is substituted with a substituted or unsubstituted C₁-C₆ alkylene,substituted or unsubstituted 2 to 6 membered heteroalkylene, substitutedor unsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to6 membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene. Inembodiments, L¹ is a bond, substituted or unsubstituted C₁-C₆ alkylene,substituted or unsubstituted 2 to 6 membered heteroalkylene, substitutedor unsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to6 membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L¹ is a substituted or unsubstituted methylene, whereinL¹ is substituted with a substituted or unsubstituted C₁-C₆ alkylene orsubstituted or unsubstituted 2 to 6 membered heteroalkylene. Inembodiments, L¹ is a substituted or unsubstituted C₁-C₆ alkylene orsubstituted or unsubstituted 2 to 6 membered heteroalkylene. Inembodiments, L¹ is a substituted or unsubstituted methylene, wherein L¹is substituted with a substituted or unsubstituted C₁-C₆ alkylene. Inembodiments, L¹ is an unsubstituted methylene.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkylene (e.g.,alkylene, alkenylene, or alkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene), substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroarylene; wherein at least one of L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) is not a bond.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₈ alkylene(e.g., alkylene, alkenylene, or alkynylene), substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 8 membered heteroalkylene(e.g., heteroalkylene, heteroalkenylene, or heteroalkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₈cycloalkylene, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 8 membered heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₆-C₁₀ arylene, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 5 to 10 memberedheteroarylene; wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) is not a bond.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₆ alkylene(e.g., alkylene, alkenylene, or alkynylene), substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkylene(e.g., heteroalkylene, heteroalkenylene, or heteroalkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkylene, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroarylene;wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D) and L^(1E) is nota bond.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalknylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₈ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) or L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkenylene (e.g.,substituted with a substituent group, or substituted with size-limitedsubstituent group), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkenylene; wherein at least one of L^(1A), L^(1B),L^(1C), L^(1D) and L^(1E) is not a bond.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) or L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₈alkenylene, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 8membered heteroalkenylene; wherein at least one of L^(1A), L^(1B),L^(1C), L^(1D) and L^(1E) is not a bond. In embodiments, L¹ isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A), L^(1B), L^(1C), L^(1D)or L^(1E) are independently a bond, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkenylene, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkenylene; wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) is not a bond.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkenylene, or substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heteroalkenylene. In embodiments, L¹is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₂-C₈ alkenylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 8 membered heteroalkenylene. Inembodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₂-C₆ alkenylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 3 to 6 memberedheteroalkenylene.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) or L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkynylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkynylene; wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) is not a bond.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) or L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₈ alkynylene, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 8membered heteroalkynylene; wherein at least one of L^(1A), L^(1B),L^(1C), L^(1D) and L^(1E) is not a bond. In embodiments, L¹ isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) are independently a bond, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkynylene, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkynylene; wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) is not a bond.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkynylene, or substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heteroalkynylene. In embodiments, L¹is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₂-C₆alkynylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 8 membered heteroalkynylene. Inembodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₂-C₆ alkynylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 3 to 6 memberedheteroalkynylene.

In embodiments, L¹ is a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkelyene (e.g., alkylene (e.g., alkylene,alkenylene, or alkynylene), alkenylene, or alkynylene) or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene). In embodiments, L¹ is an unsubstituted C₁-C₄ alkylene(e.g., alkylene, alkenylene, or alkynylene). In embodiments, L¹ is notsubstituted with a cleavable moiety. In embodiments, L¹ is notsubstituted with a monovalent cleavable moiety.

In embodiments, L¹ is a polymer. In embodiments, L² is a polymer. Inembodiments, L² includes a polymer. In embodiments, L² includes PEG. Inembodiments, L⁴ is a polymer. In embodiments, L⁴ includes a polymer. Inembodiments, L⁴ includes PEG. The term “polymer” refers to a moleculeincluding repeating subunits (e.g., polymerized monomers). For example,polymeric molecules may be based upon polyethylene glycol (PEG),tetraethylene glycol (TEG), polyvinylpyrrolidone (PVP), poly(xylene), orpoly(p-xylylene). The term “polymerizable monomer” is used in accordancewith its meaning in the art of polymer chemistry and refers to acompound that may covalently bind chemically to other monomer molecules(such as other polymerizable monomers that are the same or different) toform a polymer.

In embodiments, L² is a cleavable linker. In embodiments, L² is anon-cleavable linker. In embodiments, L² is a chemically cleavablelinker. In embodiments, L² is a photocleavable linker, an acid-cleavablelinker, a base-cleavable linker, an oxidant-cleavable linker, areductant-cleavable linker, or a fluoride-cleavable linker. Inembodiments, L² is a cleavable linker including a dialkylketal linker,an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E), and L^(2A),L^(2B), L^(2C), L^(2D), and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted or unsubstituted alkylene, substitutedor unsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; wherein at least one of L^(2A), L^(2B), L^(2C), L^(2D),and L^(2E) is not a bond.

In embodiments, L¹ is L^(2A)-L^(1B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(2B), L^(2C), L^(2D) and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₂₀alkylene,substituted or unsubstituted 2 to 20 membered heteroalkylene,substituted or unsubstituted C₃-C₂₀cycloalkylene, substituted orunsubstituted 3 to 20 membered heterocycloalkylene, substituted orunsubstituted C₆-C₂₀ arylene, or substituted or unsubstituted 5 to 20membered heteroarylene; wherein at least one of L^(2A), L^(1B), L^(2C),L^(2D), and L^(2E) is not a bond.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(1B), L^(2C), L^(2D) and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₁₀alkylene,substituted or unsubstituted 2 to 10 membered heteroalkylene,substituted or unsubstituted C₃-C₈ cycloalkylene, substituted orunsubstituted 3 to 8 membered heterocycloalkylene, substituted orunsubstituted C₆-C₁₀ arylene, or substituted or unsubstituted 5 to 10membered heteroarylene; wherein at least one of L^(2A), L^(2B), L^(2C),L^(2D), and L^(2E) is not a bond.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(2B), L^(2C), L^(2D) and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₆ alkylene,substituted or unsubstituted 2 to 6 membered heteroalkylene, substitutedor unsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to6 membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene; wherein atleast one of L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) is not a bond.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); L^(2A) is abond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene; L^(2B) is a bond, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, substitutedor unsubstituted heteroarylene; L^(2C) is a bond, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, substitutedor unsubstituted heteroarylene; L^(2D) is a bond, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene; andL^(2E) is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; wherein at least one of L^(2A), L^(2B), L^(2C), L^(2D),and L^(2E) is not a bond.

In embodiments, L² is a bond, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L² is a bond, substituted or unsubstituted C₁-C₂₀alkylene, substituted or unsubstituted 2 to 20 membered heteroalkylene,substituted or unsubstituted C₃-C₂₀cycloalkylene, substituted orunsubstituted 3 to 20 membered heterocycloalkylene, substituted orunsubstituted C₆-C₂₀ arylene, or substituted or unsubstituted 5 to 20membered heteroarylene.

In embodiments, L² is a bond, substituted or unsubstituted C₁-C₈alkylene, substituted or unsubstituted 2 to 8 membered heteroalkylene,substituted or unsubstituted C₃-C₈ cycloalkylene, substituted orunsubstituted 3 to 8 membered heterocycloalkylene, substituted orunsubstituted C₆-C₁₀ arylene, or substituted or unsubstituted 5 to 10membered heteroarylene.

In embodiments, L² is a bond, substituted or unsubstituted C₁-C₆alkylene, substituted or unsubstituted 2 to 6 membered heteroalkylene,substituted or unsubstituted C₃-C₆ cycloalkylene, substituted orunsubstituted 3 to 6 membered heterocycloalkylene. substituted orunsubstituted phenyl, or substituted or unsubstituted 5 to 6 memberedheteroarylene.

In embodiments, L² is a substituted or unsubstituted 4 to 10 memberedheteroalkylene. In embodiments, L² is a substituted or unsubstituted 4to 8 membered heteroalkylene.

In embodiments, L² is

wherein R⁵ is as described herein. In embodiments, L² is

wherein R⁵ is as described herein. In embodiments, L² is

wherein R⁵ is as described herein and ne is an integer from 0 to 20.

In embodiments, L² is

wherein R⁵ is as described herein. In embodiments, L² is

wherein R⁵ is as described herein. In embodiments, L² is

wherein R⁵ is as described herein and ne is an integer from 0 to 20.

In embodiments, ne is an integer from 0 to 18. In embodiments, ne is aninteger from 0 to 12. In embodiments, ne is an integer from 0 to 10. Inembodiments, ne is an integer from 0 to 8. In embodiments, ne is aninteger from 0 to 4. In embodiments, ne is an integer from 1 to 18. Inembodiments, ne is an integer from 1 to 12. In embodiments, ne is aninteger from 1 to 10. In embodiments, ne is an integer from 1 to 8. Inembodiments, ne is an integer from 1 to 4. In embodiments, ne is aninteger from 2 to 18. In embodiments, ne is an integer from 2 to 12. Inembodiments, ne is an integer from 2 to 10. In embodiments, ne is aninteger from 2 to 8. In embodiments, ne is an integer from 0 to 4. Inembodiments, ne is 0. In embodiments, ne is 1. In embodiments, ne is 2.In embodiments, ne is 3. In embodiments, ne is 4. In embodiments, ne is5. In embodiments, ne is 6. In embodiments, ne is 7. In embodiments, neis 8. In embodiments, ne is 9. In embodiments, ne is 10. In embodiments,ne is 11. In embodiments, ne is 12. In embodiments, ne is 13. Inembodiments, ne is 14. In embodiments, ne is 15. In embodiments, ne is16. In embodiments, ne is 17. In embodiments, ne is 18. In embodiments,ne is 19. In embodiments, ne is 20.

In embodiments, L² is —C(CH₃)₂CH₂NHC(O)—. In embodiments, L² is

In embodiments, L² includes

In embodiments, L² is a cleavable linker. In embodiments, L² is achemically cleavable linker. In embodiments, L² is a photocleavablelinker, an acid-cleavable linker, a base-cleavable linker, anoxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments, L² is a photocleavablelinker. In embodiments, L² is an acid-cleavable linker. In embodiments,L² is a base-cleavable linker. In embodiments, L² is anoxidant-cleavable linker. In embodiments, L² is a reductant-cleavablelinker. In embodiments, L² is a fluoride-cleavable linker.

In embodiments, L² includes a cleavable linker. In embodiments, L²includes a chemically cleavable linker. In embodiments, L² includes aphotocleavable linker, an acid-cleavable linker, a base-cleavablelinker, an oxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments, L² includes a photocleavablelinker. In embodiments, L² includes an acid-cleavable linker. Inembodiments, L² includes a base-cleavable linker. In embodiments, L²includes an oxidant-cleavable linker. In embodiments, L² includes areductant-cleavable linker. In embodiments, L² includes afluoride-cleavable linker.

In embodiments, L² is a cleavable linker including a dialkylketallinker, an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker. In embodiments, L² is a cleavable linker including adialkylketal linker. In embodiments, L² is a cleavable linker includingan azo linker. In embodiments, L² is a cleavable linker including anallyl linker. In embodiments, L² is a cleavable linker including acyanoethyl linker. In embodiments, L² is a cleavable linker including a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker. Inembodiments, L² is a cleavable linker including a nitrobenzyl linker.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(2B), L^(2C), L^(2D), and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(2A). L^(2B), L^(2C), L^(2D), and L^(2E) is not a bond.

In embodiments, L¹ is L^(2A)-L^(1B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(2B), L^(2C), L^(2D) and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene; wherein at leastone of L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) is not a bond.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(2B), L^(2C), L^(2D) and L^(2E) are independently a bond. —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₁₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 10 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene; wherein at leastone of L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) is not a bond.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(2B), L^(2C), L^(2D) and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene; wherein at leastone of L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) is not a bond.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E). L^(2A) is abond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene). L^(2B) is a bond, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted arylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroarylene; L^(2C) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted arylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroarylene;L^(2D) is a bond, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalknylene); and L^(2E) is a bond, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or alkynylene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted arylene,or substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroarylene; wherein at least one of L^(2A), L^(2B), L^(2C), L^(2D),and L^(2E) is not a bond.

In embodiments, L² is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene. In embodiments,L² is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₂₀alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene. In embodiments,L² is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₈ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene. In embodiments,L² is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L¹ is a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 4 to 10 membered heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene). In embodiments,L² is a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 4 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene). In embodiments, L² is asubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 4 to 6membered heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene).

In embodiments, L² is an orthogonally cleavable linker or a non-covalentlinker. In embodiments, L² includes an orthogonally cleavable linker ora non-covalent linker. In embodiments, L² is an orthogonally cleavablelinker. In embodiments, L² is a non-covalent linker.

In embodiments, L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are eachindependently

In embodiments, -L²- is

wherein z is an integer from 0 to 10. In embodiments z is an integerfrom 1 to 8. In embodiments z is an integer from 2 to 4. In embodimentsz is 0. In embodiments z is 1. In embodiments z is 2. In embodiments zis 3. In embodiments z is 4. In embodiments z is 5. In embodiments z is6. In embodiments z is 7. In embodiments z is 8. In embodiments z is 9.In embodiments z is 10.

In embodiments, L^(1A), L^(2B), L^(2C), L^(2D), and L^(2E) are eachindependently

In embodiments, L^(1A), L^(2B), L^(2C), L^(2D), and L^(2E) are eachindependently

In embodiments, L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are eachindependently

In embodiments, L^(1A), L^(2B), L^(2C), L^(1D), and L^(2E) are eachindependently

In embodiments, L^(2A), L^(2B), L^(2C), L^(2D)and L^(2E) are eachindependently

In embodiments, L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are eachindependently

In embodiments, L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are eachindependently

In embodiments, L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are eachindependently

In embodiments, L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are eachindependently

In embodiments, -L²-R⁵ is

and z is an integer from 0 to 10.

In embodiments, -L²-R⁵ is

and z is an integer from 0 to 10.

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

wherein z is an integer from 0 to 10. In embodiments z is an integerfrom 1 to 8. In embodiments z is an integer from 2 to 4. In embodimentsz is 0. In embodiments z is 1. In embodiments z is 2. In embodiments zis 3. In embodiments z is 4. In embodiments z is 5. In embodiments z is6. In embodiments z is 7. In embodiments z is 8. In embodiments z is 9.In embodiments z is 10. In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, L³ is

wherein L¹ is a bond, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene. L² is a bond,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted asubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkylene(e.g., alkylene, alkenylene, or alkynylene), substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene), substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted arylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroarylene, a cleavable linker, an orthogonallycleavable linker, non-covalent linker, or -L^(2A)-L^(2B)-L^(2C)-L^(2D)-,wherein L^(2A) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene). L^(2B) is a bond substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted arylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroarylene. L^(2C) is a bond substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted arylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroarylene.L^(2D) is a bond, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), wherein at least one of L^(2A), L^(2B), L^(2C),L^(2D) is not a bond. R^(4A) and R^(6A) are independently hydrogen, —OH,—CF, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl,—CH₂Br, —CH₂I, —CN, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R⁶ is hydrogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl,—CH₂Br, —CH₂I, —CN, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. X is a bond, O,NR^(6A), or S.

In embodiments L³ is

wherein L¹, R^(4A), X, R⁶, and L² are as described herein. Inembodiments, L³ is

wherein L² is as described herein. In embodiments, L³ is

wherein L² is as described herein.

In embodiments, L⁴ is an orthogonally cleavable linker. In embodiments,L¹ is a photocleavable linker, an acid-cleavable linker, abase-cleavable linker, an oxidant-cleavable linker, areductant-cleavable linker, or a fluoride-cleavable linker. Inembodiments, L⁴ is a cleavable linker including a dialkylketal linker,an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E); and L^(4A),L^(4B), L^(4C), L^(4D), and L^(4E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted or unsubstituted alkylene, substitutedor unsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; wherein at least one of L^(4A), L^(4B), L^(4C), L^(4D),and L^(4E) is not a bond.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E); L^(4A) is abond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene; L^(4B) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted or unsubstituted cycloalkylene,substituted or unsubstituted heterocycloalkylene, substituted orunsubstituted arylene, substituted or unsubstituted heteroarylene;L^(4C) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, substituted or unsubstitutedheteroarylene; L^(4D) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substitutedor unsubstituted alkylene, substituted or unsubstituted heteroalkylene;and L^(4E) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; wherein at least one ofL^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is a bond, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L⁴ is a substituted or unsubstituted 3 to 10 memberedheteroalkylene.

In embodiments, L⁴ is an orthogonally cleavable linker. In embodiments,L⁴ is a cleavable linker. In embodiments, L⁴ is a chemically cleavablelinker. In embodiments, L⁴ is a photocleavable linker, an acid-cleavablelinker, a base-cleavable linker, an oxidant-cleavable linker, areductant-cleavable linker, or a fluoride-cleavable linker. Inembodiments, L⁴ is a photocleavable linker. In embodiments, L⁴ is anacid-cleavable linker. In embodiments, L⁴ is a base-cleavable linker. Inembodiments, L⁴ is an oxidant-cleavable linker. In embodiments, L⁴ is areductant-cleavable linker. In embodiments, L⁴ is a fluoride-cleavablelinker. In embodiments, L⁴ is a cleavable linker including adialkylketal linker, an azo linker, an allyl linker, a cyanoethyllinker, a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker. In embodiments, L⁴ is a cleavable linker including adialkylketal linker. In embodiments, L⁴ is an azo linker. Inembodiments, L⁴ is an allyl linker. In embodiments, L⁴ is a cyanoethyllinker. In embodiments, L⁴ is a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

In embodiments, L⁴ includes an orthogonally cleavable linker. Inembodiments, L⁴ includes a cleavable linker. In embodiments, L⁴ includesa chemically cleavable linker. In embodiments, L⁴ includes aphotocleavable linker, an acid-cleavable linker, a base-cleavablelinker, an oxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments, L⁴ includes a photocleavablelinker. In embodiments, L⁴ includes an acid-cleavable linker. Inembodiments, L⁴ includes a base-cleavable linker. In embodiments, L⁴includes an oxidant-cleavable linker. In embodiments, L⁴ includes areductant-cleavable linker. In embodiments, L⁴ includes afluoride-cleavable linker. In embodiments, L⁴ includes a dialkylketallinker, an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker. In embodiments, L⁴ includes a dialkylketal linker.In embodiments, L⁴ includes an azo linker. In embodiments, L⁴ includesan allyl linker. In embodiments, L⁴ includes a cyanoethyl linker. Inembodiments, L⁴ includes a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker. Inembodiments, L⁴ includes a nitrobenzyl linker.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E), L^(4A),L^(4B), L^(4C), L^(4D), or L^(4E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E); and L^(4A),L^(4B), L^(4C), L^(4D), or L^(4E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene; wherein at leastone of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E); and L^(4A),L^(4B), L^(4C), L^(4D), or L^(4E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₁₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 10 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene; wherein at leastone of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E); and L^(4A),L^(4B), L^(4C), L^(4D), or L^(4E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene; wherein at leastone of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E); wherein L^(4A)is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or alkynylene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene); L^(4B) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; L^(4C) is abond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted arylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroarylene;L^(4D) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or alkynylene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene); and L^(4E) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene.

In embodiments, L⁴ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene.

In embodiments, L⁴ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₈ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene.

In embodiments, L⁴ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L⁴ is a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 10 membered heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene). In embodiments,L⁴ is a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene). In embodiments, L⁴ is asubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 3 to 6membered heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene).

In embodiments, L^(4z) is an orthogonally cleavable linker. Inembodiments, L^(4z) is a cleavable linker. In embodiments, L^(4z) is achemically cleavable linker. In embodiments, L^(4z) is a photocleavablelinker, an acid-cleavable linker, a base-cleavable linker, anoxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments, L^(4z) is a photocleavablelinker. In embodiments, L^(4z) is an acid-cleavable linker. Inembodiments. L^(4z) is a base-cleavable linker. In embodiments, L^(4z)is an oxidant-cleavable linker. In embodiments, L^(4z) is areductant-cleavable linker. In embodiments, L^(4z) is a cleavable linkerincluding a dialkylketal linker, an azo linker, an allyl linker, acyanoethyl linker, a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyllinker, or a nitrobenzyl linker.

In embodiments, L^(4z) includes an orthogonally cleavable linker. Inembodiments, L^(4z) includes a cleavable linker. In embodiments, L^(4z)includes a chemically cleavable linker. In embodiments, L^(4z) includesa photocleavable linker, an acid-cleavable linker, a base-cleavablelinker, an oxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments, L^(4z) includes aphotocleavable linker. In embodiments, L^(4z) includes an acid-cleavablelinker. In embodiments, L^(4z) includes a base-cleavable linker. Inembodiments, L^(4z) includes an oxidant-cleavable linker. Inembodiments, L^(4z) includes a reductant-cleavable linker. Inembodiments, L^(4z) includes a cleavable linker including a dialkylketallinker, an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE).L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4z) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) is not abond.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE); andL^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) are independently abond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene; wherein at leastone of L⁴, L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) is not abond.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE); andL^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) are independently abond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₁₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 10 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene; wherein at leastone of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) is not a bond.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE); andL^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) are independently abond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene; wherein at leastone of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) is not a bond.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE);wherein L^(4zA) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or alkynylene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene); L^(4zB) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; L^(4zC) is abond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted arylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroarylene;L^(4zD) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or alkynylene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene); and L^(4zE) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(4zA), L^(4zB), L^(4zC), L^(4zD), L^(4zE) is not a bond.

In embodiments, L^(4z) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene.

In embodiments, L^(4z) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₂₀alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene.

In embodiments, L^(4z) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₈alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene.

In embodiments, L^(4z) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ allylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L^(4z) is a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 10 membered heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalknylene). In embodiments.L^(4z) is a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene). In embodiments, L⁴ is asubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 3 to 6membered heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene).

In embodiments, L⁴ is —C(CH₃)₂CH₂NHC(O)—,

In embodiments, X is O, NR^(6A), or S. In embodiments, X is a bond. Inembodiments, X is O. In embodiments, X is NR^(A). In embodiments, X isNH. In embodiments, X is S. In embodiments, X is O, NH, or S. Inembodiments, X is not a bond.

In embodiments, R³ is —OH, monophosphate, or polyphosphate. Inembodiments. R³ is —OH. In embodiments, R³ is monophosphate. Inembodiments, R³ is polyphosphate. In embodiments, R³ is diphosphate,triphosphate, tetraphosphate, pentaphosphate, or hexaphosphate. Inembodiments, R³ is diphosphate. In embodiments, R³ is triphosphate. Inembodiments, R³ is tetraphosphate. In embodiments, R³ is pentaphosphate.In embodiments, R³ is hexaphosphate. In embodiments, R³ is triphosphateor higher polyphosphate (e.g., tetraphosphate, pentaphosphate, orhexaphosphate).

In embodiments, R^(4A) is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl. Inembodiments, R^(4A) is substituted or unsubstituted C₁-C₆ alkyl, orsubstituted or unsubstituted 2 to 6 membered heteroalkyl. Inembodiments, R^(4A) is substituted or unsubstituted C₁-C₆ alkyl. Inembodiments, R^(4A) is unsubstituted C₁-C₆ alkyl. In embodiments, R^(4A)is unsubstituted methyl.

In embodiments, R^(4A) is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

In embodiments, R^(4A) is hydrogen, CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —OH,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. In embodiments,R^(4A) is hydrogen, CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂,—CHI₂, —CH₂F, —CH₂Cl, —CH—Br, —CH₂I, —CN, —OH, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₁-C₆ alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 6 membered heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 6membered heteroaryl. In embodiments, R^(4A) is hydrogen. In embodiments,when X is a bond. R^(4A) is not hydrogen. In embodiments, when X is abond. R^(4B) is not hydrogen. In embodiments, when X is a bond, R^(4A)and R^(4B) are not hydrogen.

In embodiments, R^(4A) is hydrogen, —CH₃, CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(4A) is hydrogen, —CH₃, —CF₃, —CCl₃, —CBr₃, —Cl₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(4A) is hydrogen, —CH₃, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) C₁-C₆ alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) 3 to 6membered heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) 5 to 6 membered heteroaryl.

In embodiments, R^(4A) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl. In embodiments, R^(4A) issubstituted (e.g., substituted with a substituent group, a size-limitedsubstituent group, or lower substituent group) alkyl. In embodiments,R^(4A) is unsubstituted alkyl. In embodiments, R^(4A) is substituted orunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂). Inembodiments, R^(4A) is substituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, orC₁-C₂). In embodiments, R^(4A) is unsubstituted alkyl (e.g., C₁-C₈,C₁-C₆, C₁-C₄, or C₁-C₂).

In embodiments, R^(4A) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted heteroalkyl. In embodiments, R^(4A)is substituted (e.g., substituted with a substituent group, asize-limited substituent group, or lower substituent group) heteroalkyl.In embodiments, R^(4A) is unsubstituted heteroalkyl. In embodiments,R^(4A) is substituted or unsubstituted heteroalkyl (e.g., 2 to 8membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered). In embodiments, R^(4A) is substituted heteroalkyl (e.g., 2 to8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered). In embodiments, R^(4A) is an unsubstituted heteroalkyl (e.g.,2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4to 5 membered).

In embodiments, R^(4A) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl. In embodiments, R^(4A)is substituted (e.g., substituted with a substituent group, asize-limited substituent group, or lower substituent group) cycloalkyl.In embodiments, R^(4A) is an unsubstituted cycloalkyl. In embodiments,R^(4A) is substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,C₄-C₆, or C₅-C₆). In embodiments, R^(4A) is substituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R^(4A) isunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₁-C₆).

In embodiments, R^(4A) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl. In embodiments,R^(4A) is substituted (e.g., substituted with a substituent group, asize-limited substituent group, or lower substituent group)heterocycloalkyl. In embodiments, R^(4A) is an unsubstitutedheterocycloalkyl. In embodiments, R^(4A) is substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered). In embodiments, R^(4A)is substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,R^(4A) an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R^(4A) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted aryl. In embodiments, R^(4A) issubstituted (e.g., substituted with a substituent group, a size-limitedsubstituent group, or lower substituent group) aryl. In embodiments,R^(4A) is an unsubstituted aryl. In embodiments, R^(4A) is substitutedor unsubstituted aryl (e.g., C₆-C₁₀ or phenyl). In embodiments, R^(4A)is substituted aryl (e.g., C₆-C₁₀ or phenyl). In embodiments, R^(4A) isan unsubstituted aryl (e.g., C₆-C₁₀ or phenyl).

In embodiments, R^(4A) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted heteroaryl. In embodiments, R^(4A)is substituted (e.g., substituted with a substituent group, asize-limited substituent group, or lower substituent group) heteroaryl.In embodiments, R^(4A) is an unsubstituted heteroaryl. In embodiments,R^(4A) is substituted or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R^(4A)is substituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered). In embodiments, R^(4A) is an unsubstituted heteroaryl(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).

In embodiments, R^(4B) is hydrogen, —OH, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In embodiments, R^(4B) is—X—R⁶. In embodiments, R^(4B) is hydrogen.

In embodiments, R^(4B) is hydrogen, —CF₃, —CCl₃, —CBr₃, —Cl₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl. Inembodiments, R^(4B) is substituted or unsubstituted C₁-C₆ alkyl, orsubstituted or unsubstituted 2 to 6 membered heteroalkyl. Inembodiments, R^(4B) is substituted or unsubstituted C₁-C₆ alkyl. Inembodiments, R^(4B) is unsubstituted C₁-C₆ alkyl. In embodiments, R^(4B)is unsubstituted methyl.

In embodiments, R^(4B) is hydrogen, CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —OH,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. In embodiments,R^(4B) is hydrogen, CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂,—CHI₂, —CH₂F, —CH₂Cl, —CH—Br, —CH₂I, —CN, —OH, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₁-C₆ alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 6 membered heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 6membered heteroaryl. In embodiments, R^(4B) is hydrogen.

In embodiments, R^(4B) is hydrogen, —CH₃, CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(4B) is hydrogen, —CH₃, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(4B) is hydrogen, —CH₃, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) C₁-C₆ alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) 3 to 6membered heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) 5 to 6 membered heteroaryl.

In embodiments, R^(4B) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl. In embodiments, R^(4B) issubstituted (e.g., substituted with a substituent group, a size-limitedsubstituent group, or lower substituent group) alkyl. In embodiments,R^(4B) is unsubstituted alkyl. In embodiments, R^(4B) is substituted orunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂). Inembodiments, R^(4B) is substituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, orC₁-C₂). In embodiments. R^(4B) is unsubstituted alkyl (e.g., C₁-C₈,C₁-C₆, C₁-C₄, or C₁-C₂).

In embodiments, R^(4B) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted heteroalkyl. In embodiments, R^(4B)is substituted (e.g., substituted with a substituent group, asize-limited substituent group, or lower substituent group) heteroalkyl.In embodiments, R^(4B) is unsubstituted heteroalkyl. In embodiments,R^(4B) is substituted or unsubstituted heteroalkyl (e.g., 2 to 8membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered). In embodiments, R^(4B) is substituted heteroalkyl (e.g., 2 to8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered). In embodiments, R^(4B) is an unsubstituted heteroalkyl (e.g.,2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4to 5 membered).

In embodiments, R^(4B) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl. In embodiments, R^(4B)is substituted (e.g., substituted with a substituent group, asize-limited substituent group, or lower substituent group) cycloalkyl.In embodiments, R^(4B) is an unsubstituted cycloalkyl. In embodiments,R^(4B) is substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,C₄-C₆, or C₅-C₆). In embodiments, R^(4B) is substituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R^(4B) isunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆).

In embodiments, R^(4B) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl. In embodiments,R^(4B) is substituted (e.g., substituted with a substituent group, asize-limited substituent group, or lower substituent group)heterocycloalkyl. In embodiments, R^(4B) is an unsubstitutedheterocycloalkyl. In embodiments, R^(4B) is substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered). In embodiments, R^(4B)is substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,R^(4B) an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R^(4B) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted aryl. In embodiments, R^(4B) issubstituted (e.g., substituted with a substituent group, a size-limitedsubstituent group, or lower substituent group) aryl. In embodiments,R^(4B) is an unsubstituted aryl. In embodiments, R^(4B) is substitutedor unsubstituted aryl (e.g., C₆-C₁₀ or phenyl). In embodiments, R^(4B)is substituted aryl (e.g., C₆-C₁₀ or phenyl). In embodiments, R^(4B) isan unsubstituted aryl (e.g., C₆-C₁₀ or phenyl).

In embodiments, R^(4B) is substituted (e.g., substituted with asubstituent group, a size-limited substituent group, or lowersubstituent group) or unsubstituted heteroaryl. In embodiments, R^(4B)is substituted (e.g., substituted with a substituent group, asize-limited substituent group, or lower substituent group) heteroaryl.In embodiments, R^(4B) is an unsubstituted heteroaryl. In embodiments,R^(4B) is substituted or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R^(4B)is substituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered). In embodiments, R^(4B) is an unsubstituted heteroaryl(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).

In embodiments, R⁵ is a detectable label. In embodiments, R⁵ is afluorescent dye.

In embodiments, R⁵ is biotin, azide, trans-cyclooctene (TCO), or phenylboric acid (PBA). In embodiments, R⁵ is biotin, azide, trans-cyclooctene(TCO), phenylboronic acid (PBA), quadricyclane, or norbornene.

In embodiments, R⁵ is fluorescent dye with a molecular weight of atleast about 130 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of at least about 135 Daltons. In embodiments, R⁵ isfluorescent dye with a molecular weight of at least about 140 Daltons.In embodiments, R⁵ is fluorescent dye with a molecular weight of atleast about 145 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of at least about 150 Daltons. In embodiments, R⁵ isfluorescent dye with a molecular weight of about 130 Daltons. Inembodiments, R⁵ is fluorescent dye with a molecular weight of about 135Daltons. In embodiments, R⁵ is fluorescent dye with a molecular weightof about 140 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of about 145 Daltons. In embodiments, R⁵ is fluorescentdye with a molecular weight of about 150 Daltons. In embodiments, R⁵ isfluorescent dye with a molecular weight of about 146 Daltons.

In embodiments, R⁵ is fluorescent dye with a molecular weight of about140 to about 3000 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of about 140 to about 2500 Daltons. In embodiments, R⁵is fluorescent dye with a molecular weight of about 140 to about 2000Daltons. In embodiments, R⁵ is fluorescent dye with a molecular weightof about 140 to about 1000 Daltons. In embodiments, R⁵ is fluorescentdye with a molecular weight of about 140 to about 900 Daltons. Inembodiments, R⁵ is fluorescent dye with a molecular weight of about 140to about 800 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of about 140 to about 700 Daltons. In embodiments, R⁵is fluorescent dye with a molecular weight of about 140 to about 600Daltons. In embodiments, R⁵ is fluorescent dye with a molecular weightof about 140 to about 500 Daltons. In embodiments, R⁵ is fluorescent dyewith a molecular weight of about 140 to about 400 Daltons. Inembodiments, R⁵ is fluorescent dye with a molecular weight of about 140to about 300 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of about 140 to about 200 Daltons.

In embodiments, R⁵ is fluorescent dye with a molecular weight of about200 to about 3000 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of about 200 to about 2500 Daltons. In embodiments, R⁵is fluorescent dye with a molecular weight of about 200 to about 2000Daltons. In embodiments, R⁵ is fluorescent dye with a molecular weightof about 200 to about 1000 Daltons. In embodiments, R⁵ is fluorescentdye with a molecular weight of about 200 to about 900 Daltons. Inembodiments, R⁵ is fluorescent dye with a molecular weight of about 200to about 800 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of about 200 to about 700 Daltons. In embodiments, R⁵is fluorescent dye with a molecular weight of about 200 to about 600Daltons. In embodiments, R⁵ is fluorescent dye with a molecular weightof about 200 to about 500 Daltons. In embodiments, R⁵ is fluorescent dyewith a molecular weight of about 200 to about 400 Daltons. Inembodiments, R⁵ is fluorescent dye with a molecular weight of about 200to about 300 Daltons.

In embodiments, R⁵ is fluorescent dye with a molecular weight of about300 to about 3000 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of about 300 to about 2500 Daltons. In embodiments, R⁵is fluorescent dye with a molecular weight of about 300 to about 2000Daltons. In embodiments, R⁵ is fluorescent dye with a molecular weightof about 300 to about 1000 Daltons. In embodiments, R⁵ is fluorescentdye with a molecular weight of about 300 to about 900 Daltons. Inembodiments, R⁵ is fluorescent dye with a molecular weight of about 300to about 800 Daltons. In embodiments, R⁵ is fluorescent dye with amolecular weight of about 300 to about 700 Daltons. In embodiments, R⁵is fluorescent dye with a molecular weight of about 300 to about 600Daltons. In embodiments, R⁵ is fluorescent dye with a molecular weightof about 300 to about 500 Daltons. In embodiments, R⁵ is fluorescent dyewith a molecular weight of about 300 to about 400 Daltons.

In embodiments, R⁵ is fluorescent dye with a molecular weight of about140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090,1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210,1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330,1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450,1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570,1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690,1700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790, 1800, 1810,1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900, 1910, 1920, 1930,1940, 1950, 1960, 1970, 1980, 1990, 2000, 2010, 2020, 2030, 2040, 2050,2060, 2070, 2080, 2090, 2100, 2110, 2120, 2130, 2140, 2150, 2160, 2170,2180, 2190, 2200, 2210, 2220, 2230, 2240, 2250, 2260, 2270, 2280, 2290,2300, 2310, 2320, 2330, 2340, 2350, 2360, 2370, 2380, 2390, 2400, 2410,2420, 2430, 2440, 2450, 2460, 2470, 2480, 2490, 2500, 2510, 2520, 2530,2540, 2550, 2560, 2570, 2580, 2590, 2600, 2610, 2620, 2630, 2640, 2650,2660, 2670, 2680, 2690, 2700, 2710, 2720, 2730, 2740, 2750, 2760, 2770,2780, 2790, 2800, 2810, 2820, 2830, 2840, 2850, 2860, 2870, 2880, 2890,2900, 2910, 2920, 2930, 2940, 2950, 2960, 2970, 2980, 2990, or about3000 Daltons.

In embodiments, R⁵ is

In embodiments, R⁵ is a detectable label. In embodiments, R⁵ is afluorescent dye. In embodiments, R⁵ is an anchor moiety. In embodiments,R⁵ is a click chemistry reactant moiety. In embodiments, R⁵ is atrans-cyclooctene moiety or azide moiety. In embodiments. R⁵ is anaffinity anchor moiety. In embodiments, R⁵ is a biotin moiety. Inembodiments, R⁵ is a reactant for a bioconjugate reaction that forms acovalent bond between R⁵ and a second bioconjugate reaction reactant.

In embodiments, R⁵ is a fluorescent dye. In embodiments R⁵ is a AlexaFluor® 350 moiety, Alexa Fluor® 405 moiety, Alexa Fluor® 430 moiety,Alexa Fluor® 488 moiety, Alexa Fluor® 532 moiety, Alexa Fluor® 546moiety, Alexa Fluor® 555 moiety, Alexa Fluor® 568 moiety, Alexa Fluor®594 moiety, Alexa Fluor® 610 moiety, Alexa Fluor® 633 moiety, AlexaFluor® 635 moiety, Alexa Fluor® 647 moiety, Alexa Fluor®, 660 moiety,Alexa Fluor® 680 moiety, Alexa Fluor® 700 moiety, Alexa Fluor*) 750moiety, or Alexa Fluor® 790 moiety. In embodiments the detectable moietyis a Alexa Fluor®) 488 moiety, Rhodamine 6G (R6G) moiety, ROX ReferenceDye (ROX) moiety, or Cy5 moiety.

In embodiments R⁵ is a FAM™ moiety, TET™ moiety, JOE™ moiety, VIC®moiety, HEX™ moiety, NED™ moiety, PET® moiety, ROX™ moiety, TAMRA™moiety, TET™ moiety, Texas Red®) moiety, Alexa Fluor® 488 moiety,Rhodamine 6G (R6G) moiety, ROX Reference Dye (ROX) moiety, Sulfo-Cy5, orCy5 moiety. In embodiments R⁵ is a Rhodamine 6G (R6G) moiety, ROXReference Dye (ROX) moiety, Sulfo-Cy5, or Cy5 moiety.

In embodiments R⁵ is a FAM™ moiety. In embodiments R⁵ is a TET™ moiety.In embodiments R⁵ is a JOE™ moiety. In embodiments R⁵ is a VIC® moiety.In embodiments R⁵ is a HEX™ moiety. In embodiments R⁵ is a NED™ moiety.In embodiments R⁵ is a PET® moiety. In embodiments R⁵ is a ROX™ moiety.In embodiments R⁵ is a TAMRA™ moiety. In embodiments R⁵ is a TET™moiety. In embodiments R⁵ is a Texas Red® moiety. In embodiments R⁵ isan Alexa Fluor® 488 moiety. In embodiments R⁵ is a Rhodamine 6G (R6G)moiety. In embodiments R⁵ is a ROX Reference Dye (ROX) moiety. Inembodiments R⁵ is a Sulfo-Cy5. In embodiments R⁵ is a Cy5 moiety.

In embodiments, R⁵ is a biotin moiety. In embodiments, R⁵ is a biotinmoiety and R¹² is a streptavidin moiety.

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is —N₃. In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is unsubstituted ethynyl,

In embodiments, R⁵ is unsubstituted ethynyl.

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is or

In embodiments, R⁵ is a modified oliogonucleotide. In embodiments, R⁵ isa modified oliogonucleotide as described in Kumar et al ScientificReports (2012) 2, 684; Fuller et al, PNAS USA (2016) 113, 5233-5238; USPatent Application US20150368710, which are incorporated herein byreference for all purposes. In embodiments, R⁵ is a modifiedoliogonucleotide as observed in Example 3. In embodiments, R⁵ is amodified oliogonucleotide as observed in FIG. 40, and FIGS. 43-46.

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

wherein j1, j2, and j3 are independently an integer from 0 to 30.

In embodiments, R⁵ is

wherein j1, j2, and j3 are independently an integer from 0 to 30.

In embodiments, R⁵ is:

wherein j1 is an integer from 0 to 30.

In embodiments, R⁵ is

wherein j1, j2, and j3 are independently an integer from 0 to 30.

In embodiments, R⁶ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂,—CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl. Inembodiments, R⁶ is substituted or unsubstituted C₁-C₆ alkyl, orsubstituted or unsubstituted 2 to 6 membered heteroalkyl. Inembodiments, R⁶ is substituted or unsubstituted C₁-C₆ alkyl. Inembodiments, R⁶ is unsubstituted C₁-C₆ alkyl. In embodiments. R⁶ isunsubstituted methyl. In embodiments, R⁶ is hydrogen. In embodiments. R⁶is —CF₃. In embodiments, R⁶ is —CCl₃. In embodiments, R⁶ is —CBr₃. Inembodiments, R⁶ is —CI₃. In embodiments, R⁶ is —CHF₂. In embodiments, R⁶is —CHCl₂. In embodiments, R⁶ is —CHBr₂. In embodiments, R⁶ is —CHI₂. Inembodiments, R⁶ is —CH₂F. In embodiments, R⁶ is —CH₂Cl. In embodiments,R⁶ is —CH₂Br. In embodiments, R⁶ is —CH₂I. In embodiments, R⁶ is —CN.

In embodiments, R⁶ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂,—CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments. R⁶ is hydrogen, CF₃, —CCl₃,—CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂C₁, —CH₂Br, —CH₂I,—CN, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. In embodiments, R⁶is hydrogen.

In embodiments, R⁶ is hydrogen, —CH₃, CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN, -Ph,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R⁶ is hydrogen, —CH₃, —CF₃, —CCl₃; —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN, -Ph,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R⁶ is hydrogen. —CH₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN, -Ph,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) C₁-C₆ alkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) 2 to 6 membered heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) 3 to 6membered heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) 5 to 6 membered heteroaryl.

In embodiments, R⁶ is substituted (e.g., substituted with a substituentgroup, a size-limited substituent group, or lower substituent group) orunsubstituted alkyl. In embodiments, R⁶ is substituted (e.g.,substituted with a substituent group, a size-limited substituent group,or lower substituent group) alkyl. In embodiments, R⁶ is unsubstitutedalkyl. In embodiments, R⁶ is substituted or unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂). In embodiments. R⁶ is substituted alkyl(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂). In embodiments. R⁶ isunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂).

In embodiments, R⁶ is substituted (e.g., substituted with a substituentgroup, a size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl. In embodiments, R⁶ is substituted (e.g.,substituted with a substituent group, a size-limited substituent group,or lower substituent group) heteroalkyl. In embodiments, R⁶ isunsubstituted heteroalkyl. In embodiments, R⁶ is substituted orunsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to6 membered, 2 to 3 membered, or 4 to 5 membered). In embodiments, R⁶ issubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6membered, 2 to 3 membered, or 4 to 5 membered). In embodiments, R⁶ is anunsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to6 membered, 2 to 3 membered, or 4 to 5 membered).

In embodiments, R⁶ is substituted (e.g., substituted with a substituentgroup, a size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl. In embodiments, R⁶ is substituted (e.g.,substituted with a substituent group, a size-limited substituent group,or lower substituent group) cycloalkyl. In embodiments, R⁶ is anunsubstituted cycloalkyl. In embodiments, R⁶ is substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). Inembodiments, R⁶ is substituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, orC₅-C₆). In embodiments, R⁶ is unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, C₄-C₆, or C₅-C₆).

In embodiments, R⁶ is substituted (e.g., substituted with a substituentgroup, a size-limited substituent group, or lower substituent group) orunsubstituted heterocycloalkyl. In embodiments, R⁶ is substituted (e.g.,substituted with a substituent group, a size-limited substituent group,or lower substituent group) heterocycloalkyl. In embodiments, R⁶ is anunsubstituted heterocycloalkyl. In embodiments, R⁶ is substituted orunsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,R⁶ is substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). Inembodiments, R⁶ an unsubstituted heterocycloalkyl (e.g., 3 to 8membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6membered).

In embodiments, R⁶ is substituted (e.g., substituted with a substituentgroup, a size-limited substituent group, or lower substituent group) orunsubstituted aryl. In embodiments, R⁶ is substituted (e.g., substitutedwith a substituent group, a size-limited substituent group, or lowersubstituent group) aryl. In embodiments, R⁶ is an unsubstituted aryl. Inembodiments, R⁶ is substituted or unsubstituted aryl (e.g., C₆-C₁₀ orphenyl). In embodiments, R⁶ is substituted aryl (e.g., C₆-C₁₀ orphenyl). In embodiments, R⁶ is an unsubstituted aryl (e.g., C₆-C₁₀ orphenyl).

In embodiments, R⁶ is substituted (e.g., substituted with a substituentgroup, a size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments. R⁶ is substituted (e.g.,substituted with a substituent group, a size-limited substituent group,or lower substituent group) heteroaryl. In embodiments, R⁶ is anunsubstituted heteroaryl. In embodiments, R⁶ is substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered). In embodiments, R⁶ is substituted heteroaryl (e.g., 5 to10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R⁶ isan unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or5 to 6 membered).

In embodiments, R^(6A) is hydrogen, —OH, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl. In embodiments, R^(6A) is substituted or unsubstitutedC₁-C₆ alkyl, or substituted or unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, R^(6A) is substituted or unsubstitutedC₁-C₆ alkyl. In embodiments, R^(6A) is unsubstituted C₁-C₆ alkyl. Inembodiments, R^(6A) is unsubstituted methyl. In embodiments, R^(6A) ishydrogen. In embodiments, R^(6A) is —OH.

In embodiments, R^(6A) is hydrogen, CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —OH,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. In embodiments,R^(6A) is hydrogen, CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂,—CHI₂, —CH₂F, —CH₂Cl, —CH₂-Br, —CH₂I, —CN, —OH, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₁-C₆ alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 6 membered heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 6membered heteroaryl. In embodiments, R^(6A) is hydrogen.

In embodiments, R^(6A) is hydrogen, —CH₃, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(6A) is hydrogen, —CH₃, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(6A) is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —CN, -Ph,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) C₁-C₆ alkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) 2 to 6 membered heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) 3 to 6membered heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) 5 to 6 membered heteroaryl.

In embodiments, R⁷ is hydrogen. In embodiments, R⁷ is —OH. Inembodiments, R⁷ is —OR^(7A); and R^(7A) is hydrogen. In embodiments,R^(7A) is —OR^(1A); and R^(7A) is a polymerase-compatible moiety. Inembodiments, R⁷ is —OR^(7A); and R^(7A) is a polymerase-compatiblecleavable moiety.

In embodiments, R⁷ is —OR^(7A); and R^(7A) is a polymerase-compatiblemoiety including an azido moiety.

In embodiments, R⁷ is —OR^(7A); and R^(7A) is a polymerase-compatiblemoiety including a dithiol linker, an allyl group, an azo group, or a2-nitrobenzyl group.

In embodiments, R⁷ is —OR^(7A); and R^(7A) is a polymerase-compatiblecleavable moiety. In embodiments, R⁷ is —OR^(7A); and R^(7A) is apolymerase-compatible cleavable moiety including an azido moiety. Inembodiments, R⁷ is —OR^(7A); and R^(7A) is a polymerase-compatiblecleavable moiety including a dithiol linker, an allyl group, an azogroup, or a 2-nitrobenzyl group.

In embodiments, R^(7A) is hydrogen, polymerase-compatible moiety, orpolymerase-compatible cleavable moiety. In embodiments, R^(7A) ishydrogen. In embodiments, R^(7A) is polymerase-compatible moiety. Inembodiments, R^(7A) is a polymerase-compatible cleavable moiety. Inembodiments, R^(7A) is a polymerase-compatible cleavable moietyincluding an azido moiety. In embodiments, R^(7A) is apolymerase-compatible cleavable moiety including a dithiol linker, anallyl group, an azo group, or a 2-nitrobenzyl group. In embodiments,R^(7A) is a polymerase-compatible cleavable moiety including a dithiollinker. In embodiments, R^(7A) is a polymerase-compatible cleavablemoiety including an allyl group. In embodiments, R^(7A) is apolymerase-compatible cleavable moiety including an azo group. Inembodiments, R^(7A) is a polymerase-compatible cleavable moietyincluding a 2-nitrobenzyl group.

In embodiments, R⁷ is hydrogen. In embodiments, R⁷ is —OR^(7A); andR^(7A) is hydrogen. In embodiments, R⁷ is —OR^(7A); and R^(7A) is apolymerase-compatible cleavable moiety. In embodiments, R⁷ is —OR^(7A);and R^(7A) is a polymerase-compatible cleavable moiety including anazido moiety. In embodiments, R⁷ is —OR^(7A); and R^(7A) is apolymerase-compatible cleavable moiety including a dithiol linker. Inembodiments, R⁷ is —OR^(7A); R^(7A) is a polymerase-compatible cleavablemoiety; and the polymerase-compatible cleavable moiety is —CH₂N₃. Inembodiments, R⁷ is —OR^(7A); and R^(7A) is a polymerase-compatiblecleavable moiety comprising a dithiol linker, an allyl group, or a2-nitrobenzyl group. In embodiments, R⁷ is —NH₂, —CH₂N₃,

or —CH₂—O—CH₃.

In embodiments, R⁷ is —OR^(7A); R^(7A) is a polymerase-compatiblecleavable moiety; and the polymerase-compatible cleavable moiety is:

In embodiments, R^(7A) is

R^(8C) is hydrogen, —CX^(8C) ₃, —CHX^(8C) ₂, —CH₂X^(8C), —OCX^(8C) ₃,—OCH₂X^(8C), —OCHX^(8C) ₂, —CN, —OH, —SH, —NH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. The symbol X^(8C) is independently halogen. Inembodiments, R^(8C) is independently unsubstituted phenyl. Inembodiments, R^(8C) is —CX^(8C) ₃, —CHX^(8C) ₂, —CH₂X^(8C), —CH₂OCX^(8C)₃, —CH₂OCH₂X^(8C), —CH₂OCHX^(8C) ₂, —CN, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In embodiments, R^(8A) is independently hydrogen, —CX³ ₃, —CHX³ ₂,—CH₂X³, —OCX³ ₃, —OCH₂X³, —OCHX³ ₂, —CH₂OCX^(8C) ₃, —CH₂OCH₂X^(8C),—CH₂OCHX^(8C) ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a -substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments R^(8A) is independentlyhydrogen, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃, —OCH₂X³, —OCHX³ ₂, —CN, —OH,—SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. In embodiments,R^(8A) is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX³3, —CHX³2, —CH₂X³, —CN, or -Ph. Inembodiments, R^(8B) is independently hydrogen, deuterium. —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, or-Ph. In embodiments, R^(8A) is independently hydrogen, —CX³ ₃, —CHX³ ₂,—CH₂X³, —OCX³ ₃, —OCH₂X³, —OCHX³2, —CN, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl.

R^(8B) is independently hydrogen, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃,—OCH₂X⁴, —OCHX⁴2, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments, Ra is independently hydrogen,—CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH,—NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. In embodiments,R^(8B) is independently hydrogen, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃,—OCH₂X⁴, —OCHX⁴ ₂. —CN, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(8A) is independently hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂,—CH₂X³, —CN, -Ph, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. In embodiments.R^(8B) is independently hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(8A) and R^(8B) are independently hydrogen orunsubstituted alkyl. In embodiments, R^(8A) and R^(8B) are independentlyhydrogen or unsubstituted C₁-C₄ alkyl. In embodiments, R^(8A) and R^(8B)are independently hydrogen.

In embodiments, R⁹ is independently hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵,—OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments, R⁹ is independently hydrogen,—CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH, —SH,—NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. In embodiments, R⁹is independently hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵,—OCHX⁵ ₂, —CN, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R¹⁰ is independently hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶,—OCX⁶ ₃, —OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments, R¹⁰ is independently hydrogen,—CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH,—NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. In embodiments, R¹⁰is independently hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶,—OCHX⁶ ₂, —CN, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R⁹ is independently hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵,—OCH₃, —SCH₃, —NHCH₃, —CN, -Ph, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl; R¹⁰ isindependently hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃, —SCH₃, —NHCH₃,—CN, -Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments R¹¹ is independently hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷,—OCX⁷ ₃, —OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments, R¹¹ is independently hydrogen,—CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃, —OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH,—NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. The symbols X³, X⁴,X⁵, X⁶ and X⁷ are independently halogen. In embodiments R¹¹ isindependently hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X′, —OCX⁷ ₃, —OCH₂X⁷,—OCHX⁷ ₂, —CN, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R¹¹ is independently hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷,—OCH₃, —SCH₃, —NHCH₃, —CN, -Ph substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl.

In embodiments, R⁹, R¹⁰, and R¹¹ are independently unsubstituted alkylor unsubstituted heteroalkyl. In embodiments, R⁹, R¹⁰, and R¹¹ areindependently unsubstituted C₁-C₆ alkyl or unsubstituted 2 to 4 memberedheteroalkyl. In embodiments, R⁹, R¹⁰, and R¹¹ are independentlyunsubstituted C₁-C₆ alkyl or unsubstituted 2 to 4 membered heteroalkyl.In embodiments, R⁹, R¹⁰, and R¹¹ are independently unsubstituted methylor unsubstituted methoxy. In embodiments, R^(8A), R^(8B), R⁹, R¹⁰, andR¹¹ are independently hydrogen or unsubstituted methyl. In embodiments,R^(8A) and R^(8B) are hydrogen and R⁹, R¹⁰, and R¹¹ are unsubstitutedmethyl.

In embodiments, R⁷ is —OR^(7A); R^(7A) is a polymerase-compatiblecleavable moiety; and the polymerase-compatible cleavable moiety is:

wherein R^(8A) is hydrogen, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³, —OCH₂X³,—OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl;R^(8B) is independently hydrogen, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃,—OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl; R⁹is independently hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X³,—OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl;R¹⁰ is independently hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃,—OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl;R¹¹ is independently hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃,—OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl;and X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

In embodiments, R⁷ is —OR^(7A); R^(7A) is a polymerase-compatiblecleavable moiety; and the polymerase-compatible cleavable moiety is:

wherein R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are independently hydrogen orunsubstituted methyl. In embodiments, R⁷ is —OR^(7A); R^(7A) is apolymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is:

In embodiments, R^(7A) is hydrogen. In embodiments, R^(7A) is

In embodiments, R^(7A) is

In embodiments, R^(7A) is

In embodiments, R^(8A) is independently hydrogen, deuterium, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. Inembodiments, R^(8A) is independently hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂,—CH₂X³, —CN, -Ph, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(8A) is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

In embodiments, R^(8B) is independently hydrogen, deuterium, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃; —SCH₂CH₃, —SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. Inembodiments, R^(8B) is hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(8B) is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂. —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH, —NHCH₃, or -Ph.

In embodiments, —CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl,unsubstituted propyl, unsubstituted isopropyl, unsubstituted butyl, orunsubstituted tert-butyl.

In embodiments, R⁹ is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)s,—NHCH(CH₃)₂. —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. In embodiments, R⁹is hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R¹⁰ is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. R¹⁰ is hydrogen,—CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted aryl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroaryl;

In embodiments, R¹¹ is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. In embodiments,R¹¹ is hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCH₃, —SCH₃, —NHCH₃, —CN, -Phsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R¹² is selected from the group consisting of

a streptavidin moiety, or

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is a streptavidin moiety. In embodiments, R¹² is or

In embodiments, R¹² streptavidin, dibenzocyclooctyne (DBCO), tetrazine(TZ), or salicylhydroxamic acid (SHA).

In embodiments, R¹² is unsubstituted ethynyl,

In embodiments, R¹² is unsubstituted ethynyl. In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is

In embodiments, R¹² is or

In embodiments, R¹² is streptavidin, dibenzylcyclooctene (DBCO),tetrazine, salicylhydroxamic acid (SHA), bis(dithiobenzil)nickel(II), ornitrile oxide.

In embodiments, R¹³ is a fluorescent dye. In embodiments, R¹³ is a AlexaFluor® 350 moiety, Alexa Fluor® 405 moiety, Alexa Fluor® 430 moiety,Alexa Fluor® 488 moiety, Alexa Fluor® 532 moiety, Alexa Fluor® 546moiety, Alexa Fluor® 555 moiety, Alexa Fluor® 568 moiety, Alexa Fluor®594 moiety, Alexa Fluor® 610 moiety, Alexa Fluor® 633 moiety, AlexaFluor® 635 moiety, Alexa Fluor® 647 moiety, Alexa Fluor® 660 moiety.Alexa Fluor® 680 moiety, Alexa Fluor® 700 moiety, Alexa Fluor® 750moiety, or Alexa Fluor® 790 moiety. In embodiments the detectable moietyis a Alexa Fluor® 488 moiety, Rhodamine 6G (R6G) moiety, ROX ReferenceDye (ROX) moiety, or Cy5 moiety.

In embodiments R¹³ is a FAM™ moiety, TET™ moiety, JOE™ moiety, VIC®moiety, HEX™ moiety, NED™ moiety, PET® moiety, ROX™ moiety, TAMRA™moiety, TET™ moiety, Texas Red® moiety, Alexa Fluor® 488 moiety,Rhodamine 6G (R6G) moiety, ROX Reference Dye (ROX) moiety, Sulfo-Cy5, orCy5 moiety. In embodiments R¹³ is a Rhodamine 6G (R6G) moiety, ROXReference Dye (ROX) moiety, Sulfo-Cy5, or Cy5 moiety.

In embodiments, R¹³ is a detectable label. In embodiments, R¹³ is afluorescent dye.

In embodiments, R¹³ is fluorescent dye with a molecular weight of atleast about 130 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of at least about 135 Daltons. In embodiments, R¹³ isfluorescent dye with a molecular weight of at least about 140 Daltons.In embodiments, R¹³ is fluorescent dye with a molecular weight of atleast about 145 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of at least about 150 Daltons. In embodiments, R¹³ isfluorescent dye with a molecular weight of about 130 Daltons. Inembodiments, R¹³ is fluorescent dye with a molecular weight of about 135Daltons. In embodiments, R¹³ is fluorescent dye with a molecular weightof about 140 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of about 145 Daltons. In embodiments, R¹³ isfluorescent dye with a molecular weight of about 150 Daltons. Inembodiments, R¹³ is fluorescent dye with a molecular weight of about 146Daltons.

In embodiments, R¹³ is fluorescent dye with a molecular weight of about140 to about 3000 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of about 140 to about 2500 Daltons. In embodiments, R¹³is fluorescent dye with a molecular weight of about 140 to about 2000Daltons. In embodiments, R¹³ is fluorescent dye with a molecular weightof about 140 to about 1000 Daltons. In embodiments, R¹³ is fluorescentdye with a molecular weight of about 140 to about 900 Daltons. Inembodiments, R¹³ is fluorescent dye with a molecular weight of about 140to about 800 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of about 140 to about 700 Daltons. In embodiments, R¹³is fluorescent dye with a molecular weight of about 140 to about 600Daltons. In embodiments, R¹³ is fluorescent dye with a molecular weightof about 140 to about 500 Daltons. In embodiments, R¹³ is fluorescentdye with a molecular weight of about 140 to about 400 Daltons. Inembodiments, R¹³ is fluorescent dye with a molecular weight of about 140to about 300 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of about 140 to about 200 Daltons.

In embodiments, R¹³ is fluorescent dye with a molecular weight of about200 to about 3000 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of about 200 to about 2500 Daltons. In embodiments, R¹³is fluorescent dye with a molecular weight of about 200 to about 2000Daltons. In embodiments, R¹³ is fluorescent dye with a molecular weightof about 200 to about 1000 Daltons. In embodiments, R¹³ is fluorescentdye with a molecular weight of about 200 to about 900 Daltons. Inembodiments, R¹³ is fluorescent dye with a molecular weight of about 200to about 800 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of about 200 to about 700 Daltons. In embodiments, R¹³is fluorescent dye with a molecular weight of about 200 to about 600Daltons. In embodiments, R¹³ is fluorescent dye with a molecular weightof about 200 to about 500 Daltons. In embodiments, R¹³ is fluorescentdye with a molecular weight of about 200 to about 400 Daltons. Inembodiments, R¹³ is fluorescent dye with a molecular weight of about 200to about 300 Daltons.

In embodiments, R¹³ is fluorescent dye with a molecular weight of about300 to about 3000 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of about 300 to about 2500 Daltons. In embodiments, R¹³is fluorescent dye with a molecular weight of about 300 to about 2000Daltons. In embodiments, R¹³ is fluorescent dye with a molecular weightof about 300 to about 1000 Daltons. In embodiments, R¹³ is fluorescentdye with a molecular weight of about 300 to about 900 Daltons. Inembodiments, R¹³ is fluorescent dye with a molecular weight of about 300to about 800 Daltons. In embodiments, R¹³ is fluorescent dye with amolecular weight of about 300 to about 700 Daltons. In embodiments, R¹³is fluorescent dye with a molecular weight of about 300 to about 600Daltons. In embodiments, R¹³ is fluorescent dye with a molecular weightof about 300 to about 500 Daltons. In embodiments, R¹³ is fluorescentdye with a molecular weight of about 300 to about 400 Daltons.

In embodiments, R¹³ is fluorescent dye with a molecular weight of about100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060,1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180,1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300,1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420,1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540,1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660,1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780,1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900,1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980, 1990, 2000, 2010, 2020,2030, 2040, 2050, 2060, 2070, 2080, 2090, 2100, 2110, 2120, 2130, 2140,2150, 2160, 2170, 2180, 2190, 2200, 2210, 2220, 2230, 2240, 2250, 2260,2270, 2280, 2290, 2300, 2310, 2320, 2330, 2340, 2350, 2360, 2370, 2380,2390, 2400, 2410, 2420, 2430, 2440, 2450, 2460, 2470, 2480, 2490, 2500,2510, 2520, 2530, 2540, 2550, 2560, 2570, 2580, 2590, 2600, 2610, 2620,2630, 2640, 2650, 2660, 2670, 2680, 2690, 2700, 2710, 2720, 2730, 2740,2750, 2760, 2770, 2780, 2790, 2800, 2810, 2820, 2830, 2840, 2850, 2860,2870, 2880, 2890, 2900, 2910, 2920, 2930, 2940, 2950, 2960, 2970, 2980,2990, or about 3000 Daltons.

In embodiments, R¹³ is

In embodiments, R¹³ is a detectable label. In embodiments, R¹³ is afluorescent dye. In embodiments, R¹³ is an anchor moiety. Inembodiments, R¹³ is a click chemistry reactant moiety. In embodiments,R¹³ is a trans-cyclooctene moiety or azide moiety. In embodiments, R¹³is an affinity anchor moiety. In embodiments, R¹³ is a biotin moiety. Inembodiments, R¹³ is a reactant for a bioconjugate reaction that forms acovalent bond between R¹³ and a second bioconjugate reaction reactant.

In embodiments R¹³ is a FAM™ moiety. In embodiments R′″ is a TET™moiety. In embodiments R¹³ is a JOE™ moiety. In embodiments R¹³ is aVIC® moiety. In embodiments R¹³ is a HEX™ moiety. In embodiments R¹³ isa NED™ moiety. In embodiments R¹³ is a PET® moiety. In embodiments R¹³is a ROX™ moiety. In embodiments R¹³ is a TAMRA™ moiety. In embodimentsR¹³ is a TET™ moiety. In embodiments R¹³ is a Texas Red® moiety. Inembodiments R¹³ is an Alexa Fluor® 488 moiety. In embodiments R¹³ is aRhodamine 6G (R6G) moiety. In embodiments R¹³ is a ROX Reference Dye(ROX) moiety. In embodiments R¹³ is a Sulfo-Cy5. In embodiments R¹³ is aCy5 moiety.

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R³ is —N₃. In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is a modified oliogonucleotide. In embodiments, R¹³is a modified oliogonucleotide as described in Kumar et al ScientificReports (2012) 2, 684 Fuller et al. PNAS USA (2016) 113, 5233-5238; USPatent Application US20150368710, which are incorporated herein byreference for all purposes. In embodiments, R¹³ is a modifiedoliogonucleotide as observed in Example 3. In embodiments, R¹³ is amodified oliogonucleotide as observed in FIG. 40 and FIGS. 43-46.

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

In embodiments, R¹³ is

wherein j1, j2, and j3 are independently an integer from 0 to 30.

In embodiments, R¹³ is

wherein j1, j2, and j3 are independently an integer from 0 to 30.

In embodiments, R¹ is:

wherein j1 is an integer from 0 to 30.

In embodiments, R¹³ is

wherein j1, j2, and j3 are independently an integer from 0 to 30.

In embodiments, j1 is in an integer from 10 to 30. In embodiments, j isin an integer from 15 to 30. In embodiments, j1 is in an integer from 10to 30. In embodiments, j1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30. In embodiments, j2 is in an integer from 10 to 30. In embodiments,j2 is in an integer from 15 to 30. In embodiments, j2 is in an integerfrom 10 to 30. In embodiments, j2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30. In embodiments, j3 is in an integer from 10 to 30. Inembodiments, j3 is in an integer from 15 to 30. In embodiments, j3 is inan integer from 10 to 30. In embodiments, j3 is 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30.

In embodiments, X³ is independently —F. In embodiments, X³ isindependently —Cl. In embodiments, X³ is independently —Br. Inembodiments, X³ is independently —I. In embodiments, X⁴ is independently—F. In embodiments, X⁴ is independently —Cl. In embodiments, X⁴ isindependently —Br. In embodiments, X⁴ is independently —I. Inembodiments, X⁵ is independently —F. In embodiments, X⁵ is independently—Cl. In embodiments. X⁵ is independently —Br. In embodiments, X⁵ isindependently —I. In embodiments, X⁶ is independently —F. Inembodiments, X⁶ is independently —Cl. In embodiments, X⁶ isindependently —Br. In embodiments, X⁶ is independently —I. Inembodiments, X⁷ is independently —F. In embodiments, X⁷ is independently—Cl. In embodiments, X⁷ is independently —Br. In embodiments, X⁷ isindependently —I. In embodiments, X^(8C) is independently —F. Inembodiments, X^(8C) is independently —Cl. In embodiments, X^(8C) isindependently —Br. In embodiments, X^(8C) is independently —I.

In embodiments, z is an integer from 0 to 20. In embodiments, z is aninteger from 0 to 10. In embodiments, z is an integer from 0 to 15. Inembodiments, z is an integer from 5 to 10. In embodiments, z is 0. Inembodiments, z is 1. In embodiments, z is 2. In embodiments, z is 3. Inembodiments, z is 4. In embodiments, z is 5. In embodiments, z is 6. Inembodiments, z is 7. In embodiments, z is 8. In embodiments, z is 9. Inembodiments, z is 10. In embodiments, z is 11. In embodiments, z is 12.In embodiments, z is 13. In embodiments, z is 14. In embodiments, z is15. In embodiments, z is 16. In embodiments, z is 17. In embodiments, zis 18. In embodiments, z is 19. In embodiments, z is 20. In embodiments,m is an integer from 1 to 4. In embodiments, m is 1. In embodiments, mis 2. In embodiments, m is 3. In embodiments, m is 4.

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, R¹²-L⁴-R¹³ has the formula:

In embodiments, the nucleotide analogue has the formula:

In an aspect is provided a compound of the formula: R^(12z)-R¹⁴. R^(12z)is a complementary anchor moiety reactive group. R¹⁴ is R¹⁵-substitutedalkyl, R¹⁵-substituted heteroalkyl, R¹⁵-substituted cycloalkyl,R¹⁵-substituted heterocycloalkyl, R¹⁵-substituted aryl, orR¹⁵-substituted heteroaryl. R¹⁵ is independently R¹⁶-substituted alkyl,R¹⁶-substituted heteroalkyl, R¹⁶-substituted cycloalkyl, R¹⁶-substitutedheterocycloalkyl, R¹⁶-substituted aryl, R¹⁶-substituted heteroaryl, or adetectable dye. R¹⁶ is independently R¹⁷-substituted alkyl,R¹⁷-substituted heteroalkyl, R¹⁷-substituted cycloalkyl, R¹⁷-substitutedheterocycloalkyl, R¹⁷-substituted aryl. R¹⁷-substituted heteroaryl, or adetectable dye. R¹⁷ is independently R¹⁸-substituted alkyl,R¹⁸-substituted heteroalkyl. R¹⁸-substituted cycloalkyl, R¹⁸-substitutedheterocycloalkyl, R¹⁸-substituted aryl, R¹⁸-substituted heteroaryl, or adetectable dye. R¹⁸ is a detectable dye. In embodiments, R¹⁴ issubstituted with a plurality of R¹⁵ moieties, R¹⁵ is substituted with aplurality of R¹⁶ moieties, and R¹⁶ is substituted with a plurality ofR¹⁷ moieties.

In embodiments, R^(12z) is

a streptavidin moiety, or

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R¹² is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is a streptavidin moiety. In embodiments,R^(12z) is

in embodiments, the detectable dye is a fluorescent dye. In embodiments,the detectable dye includes a fluorescence resonance energy transferdonor fluorescent dye. In embodiments, the detectable dye includes afluorescence resonance energy transfer acceptor fluorescent dye. Inembodiments, the detectable dye includes a fluorescence resonance energytransfer donor and acceptor fluorescent dye pair connected by a linker.In embodiments, the detectable dye includes a fluorescence resonanceenergy transfer donor and acceptor fluorescent dye pair connected by alinker and separated by 0.1 nm to 10 nm.

In embodiments, the detectable dye is

In embodiments, the compound has the formula:

In embodiments, the compound has the formula:

wherein R^(12z) is as described herein.

In embodiments, R¹³ is a modified oligonucleotide, peptide, PEG,carbohydrate or a combination thereof.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np, R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np, R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np, L², and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np, R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np, L, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np, R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np, B, R⁴, X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein np and B are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B. R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B, R^(4A), X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B is as described herein.

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

In embodiments, the nucleotide analogue has the formula:

wherein B and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R⁵ is as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R⁵ is as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R⁵ is as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R⁵ is as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein L² and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), R¹², L⁴, R¹³, L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), R¹², L⁴, R¹³, L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹². L⁴, R¹³, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), R¹², L⁴, R¹³, L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R^(4A), R¹², L⁴, R¹³, L², R⁵, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B, R⁴, R¹², L⁴, R¹³, X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein B, R⁴, X, R¹², L⁴, R¹³, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹⁶, B, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, B, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, R^(4A), L², R⁵, and R⁶ are as describedherein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, R^(4A), L², R⁵, and R⁶ are as describedherein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, R^(4A), L², R⁵, and R⁶ are as describedherein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹². L⁴, R¹³, np, L², and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, R^(4A), L², R⁵, and R⁶ are as describedherein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, B, R⁴, X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, B, R⁴, X, and R⁶ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, B, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R³, np, B, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R³, np, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, and R⁵ are as described herein.

In embodiments, the nucleotide analogue has the formula:

wherein R¹², L⁴, R¹³, np, R⁵ are as described herein.

In embodiments, the nucleotide analogue is a nucleotide analoguedescribed herein (e.g., in an aspect, embodiment, example, figure,table, scheme, or claim).

III. Methods of Use

In an aspect is provided a method of incorporating a nucleotide analogueinto a nucleic acid sequence including combining a thermophilic nucleicacid polymerase, a primer hybridized to nucleic acid template, and anucleotide analogue including a detectable label, within a reactionvessel and allowing the thermophilic nucleic acid polymerase toincorporate the nucleotide analogue into the primer therebyincorporating a nucleotide analogue into a nucleic acid sequence.

In an aspect is provided a method of incorporating a nucleotide analogueinto a nucleic acid sequence including combining a nucleic acidpolymerase (e.g., thermophilic, 9° N and mutants thereof. Phi29 andmutants thereof), a primer hybridized to nucleic acid template, and anucleotide analogue including a detectable label, within a reactionvessel and allowing the thermophilic nucleic acid polymerase toincorporate the nucleotide analogue into the primer therebyincorporating a nucleotide analogue into a nucleic acid sequence.

In an aspect is provided a method for sequencing a nucleic acid,including: (i) incorporating in series with a nucleic acid polymerase(e.g., thermophilic, 9° N and mutants thereof, Phi29 and mutantsthereof), within a reaction vessel, one of four different labelednucleotide analogues into a primer to create an extension strand,wherein the primer is hybridized to the nucleic acid and wherein each ofthe four different labeled nucleotide analogues include a uniquedetectable label: (ii) detecting the unique detectable label of eachincorporated nucleotide analogue, so as to thereby identify eachincorporated nucleotide analogue in the extension strand, therebysequencing the nucleic acid, wherein each of the four different labelednucleotide analogues are of the structure formula:

wherein the first of the four different labeled nucleotide analogues, Bis a thymine or uracil hybridizing base; in the second of the fourdifferent labeled nucleotide analogues, B is an adenine hybridizingbase; in the third of the four different labeled nucleotide analogues. Bis an guanine hybridizing base; and in the fourth of the four differentlabeled nucleotide analogues, B is an cytosine hybridizing base. B is abase or analogue thereof. L¹ is covalent linker. L² is covalent linker.L⁴ is covalent linker. X is a bond, O, NR^(6A), or S. R³ is —OH,monophosphate, polyphosphate or a nucleic acid. In embodiments, R³ is atriphosphate or higher polyphosphate. R^(4A) and R^(6A) areindependently hydrogen, —OH, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂,—CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁵ is a detectable label, anchor moiety, oraffinity anchor moiety. R⁶ is hydrogen, —CF₃, —CCI₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁷ is hydrogen or —OR^(7A), wherein R^(7A) ishydrogen or a polymerase-compatible moiety. R¹² is a complementaryaffinity anchor moiety binder. R¹³ is a detectable label. The symbol“----” is a non-covalent bond. In embodiments, the nucleic acidpolymerase is a thermophilic nucleic acid polymerase. In embodiments,the nucleic acid polymerase is 9° N and mutants thereof. In embodiments,the nucleic acid polymerase is Phi29 and mutants thereof.

In another aspect is provided a method of incorporating a nucleotideanalogue into a nucleic acid sequence including combining a thermophilicnucleic acid polymerase, a primer hybridized to nucleic acid template,and a nucleotide analogue, within a reaction vessel and allowing thethermophilic nucleic acid polymerase to incorporate the nucleotideanalogue into the primer thereby incorporating a nucleotide analogueinto a nucleic acid sequence, wherein the nucleotide analogue includes afluorescent dye with a molecular weight of at least about 140 Daltons.and wherein the fluorescent dye is covalently bound at the 3′ positionof the nucleotide analogue.

In an aspect is provided a method of incorporating a nucleotide analogueinto a nucleic acid sequence comprising combining a nucleic acidpolymerase, a primer hybridized to nucleic acid template, and anucleotide analogue, within a reaction vessel and allowing said nucleicacid polymerase to incorporate said nucleotide analogue into said primerthereby incorporating a nucleotide analogue into a nucleic acidsequence, wherein said nucleotide analogue comprises a fluorescent dyewith a molecular weight of at least about 140 Daltons, wherein thefluorescent dye is covalently bound at the 3′ position of saidnucleotide analogue for sequence determination, and wherein afterremoval of the fluorescent dye by cleaving the 3′-O linker to regeneratethe 3′-OH on the DNA extension product allows continuous nucleotideanalogue incorporation and detection of multiple bases.

In embodiments, at least one of the four different labeled nucleotideanalogues is an orthogonally cleavable labeled nucleotide analogueincluding a cleavable linker (e.g., DTM), the orthogonally cleavablelabeled nucleotide analogue having the structure as described herein,and wherein the method further includes, after each of the incorporatingsteps, adding to the reaction vessel a cleaving reagent capable ofcleaving the cleavable linker (e.g., DTM). In embodiments, the cleavingreagent is an acid, base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄). In embodiments the nucleic acid sequence issingle-stranded DNA.

In embodiments, the method includes contacting the single-stranded DNA,wherein the single-stranded DNA is bound to a polymerase which is inturn attached to a membrane-embedded nanopore in an electrolytesolution, wherein the single-stranded DNA has a primer hybridized to aportion thereof, and determining the sequence of the single stranded DNAtemplate, following the steps of: (a) addition of four nucleotidesincluding 3′-O-cleavable linkers (DTM) attached with anchor moieties.The appropriate nucleotide analogue complementary to the nucleotideresidue of the single-stranded DNA (template) which is immediately 5′ toa nucleotide residue of the single-stranded DNA will be incorporated byDNA polymerase at the 3′ terminal nucleotide residue of the primer, soas to form a DNA extension product. Only a single 3′-O-anchor-cleavablelinker (DTM) nucleotide will add to the primer due to the 3′-O-beingblocked by a cleavable linker and anchor moiety, preventing furtherincorporation in this step; (b) addition to the extended primer of 4different nanopore tags attached with different binding moleculescorresponding to the 4 anchors: the appropriate binding molecule withtag will either covalently bind or complex with the 3′-O-anchornucleotide incorporated in step (a); (c) application of a voltage acrossthe membrane and measuring an electronic (ionic current) change acrossthe nanopore resulting from the tag attached thereto generated in step(b) translocating through the nanopore, wherein the electronic change isdifferent for each different type of tag, thereby identifying thenucleotide residue in the single-stranded template DNA, which iscomplementary to the incorporated tagged nucleotide; (d) cleavage of the3′-O-cleavable linker-attached tag by treatment with an appropriatecleaving agent, thus generating a free 3′-OH ready for the nextextension reaction; and (e) Iteratively performing steps (a)-(d) foreach nucleotide residue of the single-stranded DNA being sequenced,wherein in each iteration of step (a) the 3′-O-cleavable anchornucleotide is incorporated into the DNA extension product resulting fromthe previous iteration of step (d) if it is complementary to thenucleotide residue of the single-stranded (template) DNA which isimmediately 5′ to a nucleotide residue of the single-stranded DNAhybridized to the 3′ terminal nucleotide residue of the DNA extensionproduct, thereby determining the nucleotide sequence of thesingle-stranded DNA.

In embodiments, the method includes contacting the single-stranded DNAtemplate, wherein the single-strand DNA to be sequenced hybridizes tothe primer, wherein the single-stranded primer is conjugated to amembrane-embedded nanopore in an electrolyte solution, and determiningthe sequence of the single stranded DNA template, following the stepsof: (a) addition of polymerase and four nucleotides including3′-O-cleavable linkers (DTM) attached with anchor moieties. Theappropriate nucleotide analogue complementary to the nucleotide residueof the single-stranded DNA (template) which is immediately 5′ to anucleotide residue of the single-stranded DNA will be incorporated byDNA polymerase at the 3′ terminal nucleotide residue of the primer, soas to form a DNA extension product. Only a single 3′-O-anchor-cleavablelinker (DTM) nucleotide will add to the primer due to the 3′-O-beingblocked by a cleavable linker and anchor moiety, preventing furtherincorporation in this step: (b) addition to the extended primer of 4different nanopore tags attached with different binding moleculescorresponding to the 4 anchors; the appropriate binding molecule withtag will either covalently bind or complex with the 3′-O-anchornucleotide incorporated in step (a); (c) application of a voltage acrossthe membrane and measuring an electronic (ionic current) change acrossthe nanopore resulting from the tag attached thereto generated in step(b) translocating through the nanopore, wherein the electronic change isdifferent for each different type of tag, thereby identifying thenucleotide residue in the single-stranded template DNA, which iscomplementary to the incorporated tagged nucleotide; (d) cleavage of the3′-O-cleavable linker-attached tag by treatment with an appropriatecleaving agent, thus generating a free 3′-OH ready for the nextextension reaction; and (e) Iteratively performing steps (a)-(d) foreach nucleotide residue of the single-stranded DNA being sequenced,wherein in each iteration of step (a) the 3′-O-cleavable anchornucleotide is incorporated into the DNA extension product resulting fromthe previous iteration of step (d) if it is complementary to thenucleotide residue of the single-stranded (template) DNA which isimmediately 5′ to a nucleotide residue of the single-stranded DNAhybridized to the 3′ terminal nucleotide residue of the DNA extensionproduct, thereby determining the nucleotide sequence of thesingle-stranded DNA.

In embodiments, the method includes sequencing nucleic acid including:a) providing a nucleic acid template hybridized to a primer; b)extending the primer hybridized to the nucleic acid template with alabeled nucleotide or nucleotide analog, wherein the labeled nucleotideor nucleotide analog includes nucleotide analogs with a label linked tothe base and a blocking group on the 3′-hydroxyl group, and nucleotidesor nucleotide analogs with a cleavable label blocking the 3′ OH; and c)identifying the labeled nucleotide, so as to sequence the nucleic acid.In embodiments, the nucleic acid polymerase is a thermophilic nucleicacid polymerase. In embodiments, the nucleic acid polymerase is 9° N andmutants thereof. In embodiments, the nucleic acid polymerase is Phi29and mutants thereof.

In embodiments, at least four of the nucleotide analogues (e.g.,3′-O-Anchor-Cleavable Linker nucleotides) include a triphosphate or apolyphosphate, a base which is adenine, guanine, cytosine, thymine, oruracil, or a derivative of each thereof, and an anchor moleculecovalently coupled to the 3′-O-position of the nucleotide sugar moietyincluding a cleavable linker at the 3′-O-position.

In embodiments, the method includes simultaneously sequencing aplurality of different nucleic acids, including: a) extending aplurality of priming DNA strands hybridized to template DNAs, each ofwhich includes one of the priming DNA strands, by incorporating alabeled nucleotide; and b) identifying each labeled nucleotide, so as tosimultaneously sequence the plurality of different nucleic acids.

In embodiments, R⁵ is anchor moiety, the method further including, afterthe incorporating, labeling the nucleotide analog with a detectablelabel. In embodiments, R⁵ is an affinity anchor moiety. In embodiments,the labeling includes adding to the reaction vessel a compound havingthe formula R¹²-L⁴-R¹³, wherein R¹² is a complementary affinity anchormoiety binder; R¹³ is a detectable label; and L⁴ is a covalent linker.

In embodiments, R⁵ is a chemically reactive anchor moiety. Inembodiments, R⁵ is a bioconjugate reactive group.

In embodiments, the labeling includes adding to the reaction vessel acompound having the formula R^(12z)-L⁴-R¹³, wherein R^(12z) is acomplementary anchor moiety reactive group; R¹³ is a detectable label;and L^(4z) is a covalent linker. In embodiments, R^(12z)-L^(4z)-R¹³ hasthe structure as described herein. In embodiments, L^(4z) is a cleavablelinker.

In embodiments, the method further including, after the incorporating,cleaving the cleavable linker (e.g., DTM) with a cleaving reagent. Inembodiments, the cleaving reagent is an acid, base, oxidizing agent,reducing agent, Pd(0), tris-(2-carboxyethyl)phosphine, dilute nitrousacid, fluoride, tris(3-hydroxypropyl)phosphine), sodium dithionite(Na₂S₂O₄), or hydrazine (N₂H₄).

In embodiments, the method forms part of a sequencing by synthesismethod. In embodiments, the nucleotide analogue is3′-O-Alexa488-t-Butyldithiomethiomethyl-dCTP,3′-O-Cy5-t-Butyldithiomethyl-dGTP, 3′-O-Rox-t-Butyldithiomethyl-dATP,3′-O-RG6-t-Butyldithiomethyl-dTTP,3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP,3′-O-RG6-PEG₄-t-Butyldithiomethyl-dTTP,3′-O-Rox-PEG₄-t-Butyldithiomethyl-dATP, or3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP.

In embodiments, the thermophilic nucleic acid polymerase is a Taqpolymerase, Therminator γ, 9° N polymerase (exo-), Therminator II,Therminator III, or Therminator IX. In embodiments, the thermophilicnucleic acid polymerase is Therminator γ. In embodiments, thethermophilic nucleic acid polymerase is 9° N polymerase (exo-). Inembodiments, the thermophilic nucleic acid polymerase is Therminator II.In embodiments, the thermophilic nucleic acid polymerase is TherminatorIII. In embodiments, the thermophilic nucleic acid polymerase isTherminator IX. In embodiments, the thermophilic nucleic acid polymeraseis a Taq polymerase. In embodiments, the nucleic acid polymerase is athermophilic nucleic acid polymerase. In embodiments, the nucleic acidpolymerase is 9° N and mutants thereof. In embodiments, the nucleic acidpolymerase is Phi29 and mutants thereof. In embodiments, the polymeraseis a non-thermophilic nucleic acid polymerase.

In embodiments, the method is a method described in a figure andcorresponding figure description (e.g., FIGS. 3A-3B. FIGS. 13A-13B,FIGS. 16A-16C, FIGS. 18A-18C, FIGS. 20A-20C, FIG. 22, FIG. 47, FIGS.48A-48B. FIGS. 49A-49B, FIGS. 50A-50B, FIGS. 51A-51B. FIGS. 52A-52C,FIG. 74, FIG. 75, FIG. 76, FIG. 77, FIG. 78, FIG. 79, or FIG. 80).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EMBODIMENTS

Terms defined herein refer only to aspects and embodiments within this“Embodiments” section.

For the embodiments below, each embodiment disclosed herein iscontemplated as being applicable to each of the other disclosedembodiments. In addition, the elements recited in the compoundembodiments can be used in the composition and method embodimentsdescribed herein and vice versa.

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below. A=Adenine; C=Cytosine;G=Guanine; T=Thymine; U=Uracil; DNA=Deoxyribonucleic acid;RNA=Ribonucleic acid; “Nucleic acid” shall mean, unless otherwisespecified, any nucleic acid molecule, including, without limitation,DNA, RNA and hybrids thereof. In an embodiment the nucleic acid basesthat form nucleic acid molecules can be the bases A, C, G, T and U, aswell as derivatives thereof. Derivatives or analogs (also referred toherein as analogues) of these bases are well known in the art, and areexemplified in PCR Systems, Reagents and Consumables (Perkin ElmerCatalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J.,USA).

A “nucleotide residue” is a single nucleotide in the state it existsafter being incorporated into, and thereby becoming a monomer of, apolynucleotide. Thus, a nucleotide residue is a nucleotide monomer of apolynucleotide, e.g. DNA, which is bound to an adjacent nucleotidemonomer of the polynucleotide through a phosphodiester bond at the 3′position of its sugar and is bound to a second adjacent nucleotidemonomer through its phosphate group, with the exceptions that (i) a 3′terminal nucleotide residue is only bound to one adjacent nucleotidemonomer of the polynucleotide by a phosphodiester bond from itsphosphate group, and (ii) a 5′ terminal nucleotide residue is only boundto one adjacent nucleotide monomer of the polynucleotide by aphosphodiester bond from the 3′ position of its sugar.

“Substrate” or “Surface” shall mean any suitable medium present in thesolid phase to which a nucleic acid or an agent may be affixed.Non-limiting examples include chips, beads, nanopore structures andcolumns. In an embodiment the solid substrate can be present in asolution, including an aqueous solution, a gel, or a fluid.

“Hybridize” shall mean the annealing of one single-stranded nucleic acidto another nucleic acid based on the well-understood principle ofsequence complementarity. In an embodiment the other nucleic acid is asingle-stranded nucleic acid. The propensity for hybridization betweennucleic acids depends on the temperature and ionic strength of theirmilieu, the length of the nucleic acids and the degree ofcomplementarity. The effect of these parameters on hybridization is wellknown in the art (see Sambrook J, Fritsch E F, Maniatis T, 1989.Molecular cloning: a laboratory manual. Cold Spring Harbor LaboratoryPress, New York.). As used herein, hybridization of a primer sequence,or of a DNA extension product, to another nucleic acid shall meanannealing sufficient such that the primer, or DNA extension product,respectively, is extendable by creation of a phosphodiester bond with anavailable nucleotide or nucleotide analog capable of forming aphosphodiester bond.

As used herein, unless otherwise specified, a base which is “unique” or“different from” another base or a recited list of bases shall mean thatthe base has a different structure from the other base or bases. Forexample, a base that is “unique” or “different from” adenine, thymine,and cytosine would include a base that is guanine or a base that isuracil.

As used herein, unless otherwise specified, “primer” means anoligonucleotide that upon forming a duplex with a polynucleotidetemplate, is capable of acting as a point of polymerase incorporationand extension from its 3′ end along the template, thereby resulting inan extended duplex.

As used herein, unless otherwise specified, a label or tag moiety whichis different from the label or tag moiety of a referenced molecule meansthat the label or tag moiety has a different chemical structure from thechemical structure of the other/referenced label or tag moiety.

In some embodiments of the invention, vibrational spectroscopy is usedto detect the presence of incorporated nucleotide analogs. Vibrationalspectroscopy is a spectrographic analysis where the sample isilluminated with incident radiation in order to excite molecularvibrations. Vibrational excitation, caused by molecules of the sampleabsorbing, reflecting or scattering a particular discrete amount ofenergy, is detected and can be measured. The two major types ofvibrational spectroscopy are infrared (usually FTIR) and Raman. If FTIRis employed, then the IR spectra of the nucleotide analogs are measured.If Raman is employed, then the Raman spectra of the nucleotide analogsis measured (for example of the nucleotide analogs and in the methodsdescribed herein). These methods are disclosed in Patent Applications20150080232 and 20160024570 (Ju et al).

In certain embodiments, the polymerase, single-stranded polynucleotide,RNA, or primer is bound to a solid substrate via 1,3-dipolarazide-alkyne cycloaddition chemistry. In an embodiment the polymerase,DNA, RNA, or primer, is bound to the solid substrate via a polyethyleneglycol molecule. In an embodiment the polymerase, DNA, RNA, primer, orprobe is alkyne-labeled. In an embodiment the polymerase, DNA, RNA,primer, or probe is bound to the solid substrate via a polyethyleneglycol molecule and the solid substrate is azide-functionalized. In anembodiment the polymerase, DNA, RNA, or primer, is immobilized on thesolid substrate via an azido linkage, an alkynyl linkage, orbiotin-streptavidin interaction. Immobilization of nucleic acids isdescribed in Immobilization of DNA on Chips II, edited by ChristineWittmann (2005), Springer Verlag, Berlin, which is hereby incorporatedby reference. In an embodiment the DNA is single-strandedpolynucleotide. In an embodiment the RNA is single-stranded RNA.

In other embodiments, the solid substrate is in the form of a chip, abead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, aporous media, a porous nanotube, or a column. This invention alsoprovides the instant method, wherein the solid substrate is a metal,gold, silver, quartz, silica, a plastic, polypropylene, a glass, ordiamond. This invention also provides the instant method, wherein thesolid substrate is a porous non-metal substance to which is attached orimpregnated a metal or combination of metals. The solid surface may bein different forms including the non-limiting examples of a chip, abead, a tube, a matrix, a nanotube. The solid surface may be made frommaterials common for DNA microarrays, including the non-limitingexamples of glass or nylon. The solid surface, for examplebeads/micro-beads, may be in turn immobilized to another solid surfacesuch as a chip.

In one embodiment, the surface or substrate is a SERS-prepared surfaceor substrate designed specifically for detection of a label nucleotide.The surface may include one or more nanoplasmonic antenna, wherein thenanoplasmonic antenna may be a nanoplasmonic bowtie antenna. In oneembodiment, the nanoplasmonic bowtie antenna comprises crossed-bowtiestructure in which one pair of triangles couples to incident field,while another pair of triangles couples to Raman scattered field in anorthogonal polarization. It is also contemplated that the nanoplasmonicantenna may be an array of antennas. In addition, the nanoplasmonicantenna may include DNA functionalized sites, and may have a gap sizerange from 50 nm to 1 nm. In another embodiment, a nucleotide polymeraseis immobilized within the gap.

In another embodiment the nucleotide polymerase SERS-prepared anddesigned specifically for detection of a labeled nucleotide and/ornucleoside. The surface may include one or more nanoplasmonic antenna,wherein the nanoplasmonic antenna may be a nanoplasmonic bowtie antenna.In one embodiment, the nanoplasmonic bowtie antenna comprisescrossed-bowtie structure in which one pair of triangles couples toincident field, while another pair of triangles couples to Ramanscattered field in an orthogonal polarization. It is also contemplatedthat the nanoplasmonic antenna may be an array of antennas. In addition,the nanoplasmonic antenna may have a gap size range from 12 nm to 1 nm.In another embodiment, a nucleotide polymerase is immobilized within ona surface, substrate, or nanoplasmonic antenna on a surface.

In another embodiment, the surface comprises a DNA origami scaffold oran array of DNA origami scaffolds. It is also contemplated that the DNAorigami scaffold further comprising a primer molecules positionedbetween Au and Ag nanoparticles and nanorods located at specifiedbinding sites.

In a further embodiment, the surface comprises plasmonic crystals or anarray of plasmonic structures. For example, the plasmonic structures maybe periodic TiO—Au—TiO structures.

In various embodiments the polymerase, nucleic acid samples, DNA, RNA,primer, or probe are separated in discrete compartments, wells ordepressions on a surface.

In this invention methods are provided wherein about 1000 or fewercopies of the polymerase, nucleic acid sample, DNA, RNA, or primer arebound to the substrate. This invention also provides the instant methodswherein 2×10⁷, 1×10⁷, 1×10⁶ or 1×10⁴ or fewer copies of the polymerase,nucleic acid sample, DNA, RNA, or primer are bound to the substrate orsurface.

In some embodiments, the immobilized polymerase, nucleic acid sample,DNA, RNA, or primer, is immobilized at a high density. This inventionalso provides the instant methods wherein over or up to 1×10′, 1×10⁸,1×10⁹ copies of the polymerase, nucleic acid sample. DNA, RNA, or primerare bound to the substrate or surface.

In other embodiments of the methods and/or compositions of thisinvention, the DNA is single-stranded. In other embodiments of themethods or of the compositions described herein, the single-strandedpolynucleotide is replaced with an RNA that is single-stranded.

Because of well-understood base-pairing rules, determining thewavenumber of the Raman spectroscopy peak of a dNTP analog incorporatedinto a primer or DNA extension product, and thereby the identity of thedNTP analog that was incorporated, permits identification of thecomplementary nucleotide residue in the single-stranded polynucleotidethat the primer or DNA extension product is hybridized to. Thus, if thedNTP analog that was incorporated has a unique wavenumber in the Ramanspectroscopy peak identifying it as comprising an adenine, a thymine, acytosine, or a guanine, then the complementary nucleotide residue in thesingle-stranded polynucleotide is identified as a thymine, an adenine, aguanine or a cytosine, respectively. The purine adenine (A) pairs withthe pyrimidine thymine (T). The pyrimidine cytosine (C) pairs with thepurine guanine (G). Similarly, with regard to RNA, if the dNTP analogthat was incorporated comprises an adenine, a uracil, a cytosine, or aguanine, then the complementary nucleotide residue in thesingle-stranded RNA is identified as a uracil, an adenine, a guanine ora cytosine, respectively.

Incorporation into an oligonucleotide or polynucleotide (such as aprimer or DNA extension strand) of a nucleotide and/or nucleoside analogmeans the formation of a phosphodiester bond between the 3′ carbon atomof the 3′ terminal nucleotide residue of the polynucleotide and the 5′carbon atom of the dNTP analog resulting in the loss of pyrophosphatefrom the dNTP analog.

A Raman spectroscopy system, as can be used in the methods describedherein, typically comprises an excitation source (such as a laser,including a laser diode in appropriate configuration, or two or morelasers), a sample illumination system and light collection optics, awavelength selector (such as a filter or spectrophotometer), and adetection apparatus (such as a CCD, a photodiode array, or aphotomultiplier). Interference (notch) filters with cut-off spectralrange of ±80-120 cm⁻¹ from the laser line can be used for stray lightelimination. Holographic gratings can be used. Double and triplespectrometers allow taking Raman spectra without use of notch filters.Photodiode Arrays (PDA) or a Charge-Coupled Devices (CCD) can be used todetect Raman scattered light.

In an embodiment, surface enhanced Raman spectroscopy (SERS) is usedwhich employs a surface treated with one or more of certain metals knownin the art to cause SERS effects. In an embodiment the surface is asurface to which the polymerase, polynucleotide, single-strandedpolynucleotide, single-stranded DNA polynucleotide, single-stranded RNA,primer, DNA extension strand, or oligonucleotide probe of the methodsdescribed herein is attached. Many suitable metals are known in the art.In an embodiment the surface is electrochemically etched silver ortreated with/comprises silver and/or gold colloids with average particlesize below 20 nm. The wavenumber of the Raman spectroscopy peak of anentity is identified by irradiating the entity with the excitationsource, such as a laser, and collecting the resulting Raman spectrumusing a detection apparatus. The wavenumber of the Raman spectroscopypeak is determined from the Raman spectrum. In an embodiment, thespectrum measured is from 2000 cm⁻¹ to 2300 cm⁻¹ and the wavenumber ofthe Raman spectroscopy peak is the peak wavenumber within that spectrum.In an embodiment the spectrum measured is a sub-range of 2000 cm⁻¹ to2300 cm⁻¹ and the Raman spectroscopy peak wavenumber is the peakwavenumber within that spectrum sub-range.

Where a range of values is provided, unless the context clearly dictatesotherwise, it is understood that each intervening integer of the value,and each tenth of each intervening integer of the value, unless thecontext clearly dictates otherwise, between the upper and lower limit ofthat range, and any other stated or intervening value in that statedrange, is encompassed within the invention. The upper and lower limitsof these smaller ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding (i) either or (ii)both of those included limits are also included in the invention.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and may be unsubstituted or substituted. Thus, C1-Cn as in“C1-Cn alkyl” includes groups having 1, 2, . . . , n−1 or n carbons in alinear or branched arrangement. For example, a “C1-C5 alkyl” includesgroups having 1, 2, 3, 4, or 5 carbons in a linear or branchedarrangement, and specifically includes methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, and pentyl.

As used herein, “alkenyl” refers to a non-aromatic hydrocarbon group,straight or branched, containing at least 1 carbon to carbon doublebond, and up to the maximum possible number of non-aromaticcarbon-carbon double bonds may be present, and may be unsubstituted orsubstituted. For example, “C₂-C₅ alkenyl” means an alkenyl group having2, 3, 4, or 5, carbon atoms, and up to 1, 2, 3, or 4, carbon-carbondouble bonds respectively. Alkenyl groups include ethenyl, propenyl, andbutenyl.

The term “alkynyl” refers to a hydrocarbon group straight or branched,containing at least 1 carbon to carbon triple bond, and up to themaximum possible number of non-aromatic carbon-carbon triple bonds maybe present, and may be unsubstituted or substituted. Thus, “C₂-C₅alkynyl” means an alkynyl group having 2 or 3 carbon atoms and 1carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl andbutynyl.

The term “substituted” refers to a functional group as described abovesuch as an alkyl, or a hydrocarbyl, in which at least one bond to ahydrogen atom contained therein is replaced by a bond to non-hydrogen ornon-carbon atom, provided that normal valencies are maintained and thatthe substitution(s) result(s) in a stable compound. Substituted groupsalso include groups in which one or more bonds to a carbon(s) orhydrogen(s) atom are replaced by one or more bonds, including double ortriple bonds, to a heteroatom. Non-limiting examples of substituentsinclude the functional groups described above, and for example, N, e.g.so as to form —CN.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

In choosing the compounds of the present invention, one of ordinaryskill in the art will recognize that the various substituents, i.e. R₁,R₂, etc. are to be chosen in conformity with well-known principles ofchemical structure connectivity.

In the compound structures depicted herein, hydrogen atoms, except onribose and deoxyribose sugars, are generally not shown. However, it isunderstood that sufficient hydrogen atoms exist on the representedcarbon atoms to satisfy the octet rule.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by a one of ordinary skillin the art to which this invention belongs.

As used herein, unless otherwise stated, the singular forms ‘a’, ‘an’,and ‘the’ include plural referents unless the context clearly dictatesotherwise. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as ‘solely’,‘only’ and the like in connection with the recitation of claim elements,or use of a ‘negative limitation’.

As used herein “anchor” refers to a small chemical moiety thatorthogonally and rapidly reacts with another chemical group that carriesa detectable label. As used herein, unless otherwise specified, the“cleavable group” refers to a small chemical moiety that can be cleavedby either chemical or photochemical means.

As used herein, unless otherwise specified, a label or tag moiety whichis “different” from the label or tag moiety of a referenced moleculemeans that the label or tag moiety has a different chemical structurefrom the chemical structure of the other/referenced label or tag moiety.

All combinations of the various elements described herein are within thescope of the invention. All sub-combinations of the various elementsdescribed herein are also within the scope of the invention.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

The invention provides for a nucleotide analog consisting of (i) a base,(ii) a sugar which may be a deoxyribose or a ribose, (iii) at-butyldithiomethyl linker bound to the 3′-oxygen of the deoxyribose orribose, and (iv) a detectable label bound to the t-butyldithiomethyllinker.

The invention also provides for a method for determining the identity ofa nucleotide at a predetermined position in a nucleic acid of interest,comprising:

-   -   a) providing        -   1) the nucleic acid of interest,        -   2) a nucleic acid polymerase,        -   3) a primer capable of hybridizing to said nucleic acid            immediately 3′ of such predetermined position,        -   4) four different nucleotide analogs of claim 1, each of            which consists of one of adenine or an analog of adenine,            guanine or an analog of guanine, cytosine or an analog of            cytosine, thiamine or an analog of thiamine, and a unique            detectable label:    -   b) incorporating one of said nucleotide analogs onto the end of        said primer to form an extension strand;    -   c) detecting the unique detectable label of the incorporated        nucleotide analog so as to thereby identify the incorporated        nucleotide analog on the end of said extension strand; and    -   d) based on the identity of the incorporated nucleotide,        determining the identity of the nucleotide at the predetermined        position.

The invention also provides for A process for producing a3′-O-Bodipy-t-Butyldithiomethyl-dNTP, comprising:

-   -   a) reacting,        -   1) a 5′-O-tert-Butyldimethylsilyl-nucleoside,        -   2) acetic acid,        -   3) acetic anhydride, and        -   4) DMSO under conditions permitting the production of a            3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside;    -   b) contacting the        3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside        produced in step a) with trimethylamine, molecular sieve,        sulfuryl chloride, potassium p-toluenethiosulfonate, and        2,2,2,-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide, under        conditions permitting the production of a product having the        structure:

wherein B is a nucleobase:

-   -   c) contacting the product produced in step b) with        tetrabutylammonium fluoride THF solution under conditions        permitting the production of a product having the structure:

-   -   -   wherein B is a nucleobase;

    -   d) contacting the product produced in step c) with        tetrabutylammonium pyrophosphate,        2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one, iodine solution        and ammonium hydroxide under conditions permitting the        production of a 3-O-NH₂-t-Butyldithiomethyl-dNTP;

    -   e) contacting the 3-O-NH₂-t-Butyldithiomethyl-dNTP of step d)        with Bodipy FL-NHS ester under conditions permitting the        production of the 3′-O-Bodipy-t-Butyldithiomethyl-dNTP.

The invention also provides for a process for producing a3′-O-Bodipy-PEG₄-t-Butyldithiomethyl-dNTP, comprising:

-   -   a) reacting,        -   1) a 5′-O-tert-Butyldimethylsilyl-nucleoside,        -   2) acetic acid,        -   3) acetic anhydride, and        -   4) DMSO            under conditions permitting the production of a            3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside;    -   b) contacting the        3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside        produced in part a) with trimethylamine, molecular sieve,        sulfuryl chloride, potassium p-toluenethiosulfonate, and        2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide, under        conditions permitting the production of a product having the        structure:

wherein B is a nucleobase;

-   -   c) contacting the product produced in step b) with        tetrabutylammonium fluoride THF solution under conditions        permitting the production of a product having the structure:

-   -   -   wherein B is a nucleobase;

    -   d) contacting the product produced in step c) with        tetrabutylammonium pyrophosphate,        2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one, iodine solution        and ammonium hydroxide under conditions permitting the        production of a 3-O-NH₂-t-Butyldithiomethyl-dNTP;

    -   e) contacting the 3-O-NH₂-1-Butyldithiomethyl-dNTP of step d)        with Bodipy-PEG₄-Acid, N,N-disuccinimidyl carbonate, and        4-dimethylaminopyridine under conditions permitting the        production of the 3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dNTP.

The invention also provides for a process for producing a3′-O-Rox-t-Butyldithiomethyl-dATP, comprising:

-   -   a) reacting,        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine,            and        -   2) acetic acid and acetic anhydride, and        -   3) DMSO            under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution, and ammonium hydroxide under        conditions permitting the production of        3′-O-NH₂-t-Butyldithiomethyl-dATP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dATP produced in        step d) with ROX-NHS ester under conditions permitting the        production of the 3′-O-Rox-t-Butyldithiomethyl-dATP.

The invention also provides for a process for producing a3′-O-Rox-PEG₄-t-Butyldithiomethyl-dATP, comprising:

-   -   a) reacting,        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine,            and        -   2) acetic acid and acetic anhydride; and        -   3) DMSO            under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine:    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, I-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of        3′-O-NH₂-t-Butyldithiomethyl-dATP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dATP produced in        step d) with ROX-PEG₄-Acid, N,N-disuccinimidyl carbonate, and        4-dimethylaminopyridine under conditions permitting the        production of the 3′-O-Rox-PEG₄-Butyldithiomethyl-dATP.

The invention also provides for a process for producing a3′-O-Alexa488-t-Butyldithiomethyl-dCTP, comprising:

-   -   a) reacting        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine,            and        -   2) acetic acid and acetic anhydride, and        -   3) DMSO            under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of a        3′-O-NH₂-t-Butyldithiomethyl-dCTP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dCTP produced in        step d) with Alexa488-NHS ester under conditions permitting the        production of the 3′-O-Alexa488-t-Butyldithiomethyl-dCTP.

The invention also provides for a process for producing a3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP, comprising:

-   -   a) reacting        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine,            and        -   2) acetic acid and acetic anhydride, and        -   3) DMSO            under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine:    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of a        3′-O-NH₂-t-Butyldithiomethyl-dCTP;    -   e) contacting the 3′-O-NH₂-1-Butyldithiomethyl-dCTP produced in        step d) with Alexa488-PEG₄-NHS ester, N,N-disuccinimidyl        carbonate, and 4-dimethylaminopyridine under conditions        permitting the production of the        3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP.

The invention also provides for a process for producing a3′-O-Cy5-t-Butyldithiomethyl-dGTP, comprising:

-   -   a) reacting        -   1) a 2′-deoxyguanosine, and        -   2) tert-butyldimethylsilyl chloride, imidazole, and            N,N-dimethylformamide dimethyl acetal,            under conditions permitting the formation of a            N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine;    -   b) contacting the        N-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine produced in        step a) with acetic acid, acetic anhydride, and DMSO under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with trimethylamine,        molecular sieves, sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   d) contacting the product of step c) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   e) contacting product of step d) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of        3′-O-NH₂-t-Butyldithiomethyl-dGTP;    -   1) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dGTP produced in        step e) with Cy5-NHS under conditions permitting the production        of the 3′-O-Cy5-t-Butyldithiomethyl-dGTP.

The invention also provides for a process for producing a3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP, comprising:

-   -   a) reacting        -   1) a 2′-deoxyguanosine, and        -   2) tert-butyldimethylsilyl chloride, imidazole, and            N,N-dimethylformamide dimethyl acetal,            under conditions permitting the formation of a            N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine;    -   b) contacting the        N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine produced        in step a) with acetic acid, acetic anhydride, and DMSO under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with trimethylamine,        molecular sieves, sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   d) contacting the product of step c) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   e) contacting product of step d) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of        3′-O-NH₂-t-Butyldithiomethyl-dGTP;        contacting the 3′-O-NH₂-t-Butyldithiomethyl-dGTP produced in        step e) with Cy5-PEG₄-NHS under conditions permitting the        production of the 3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP.

The invention provides for a nucleotide analog consisting of (i) a base,(ii) a sugar which may be a deoxyribose or a ribose, (iii) at-butldithiomethyl linker bound to the 3′-oxygen of the deoxyribose orribose, and (iv) a detectable label bound to the t-butyldithiomethyllinker.

In an embodiment, the sugar is a deoxyribose. In an embodiment, thesugar is a ribose. In an embodiment, the nucleotide analog is anucleotide monophosphate, a nucleotide diphosphate, a nucleotidetriphosphate, a nucleotide tetraphosphate, a nucleotide pentaphosphate,or a nucleotide hexaphosphate. In an embodiment, the base is adenine oran analog of adenine, guanine or an analog of guanine, cytosine or ananalog of cytosine, thymine or an analog of thymine or uracil or ananalog of uracil.

In an embodiment, the t-butyldithiomethyl linker has the structure:

wherein α represents the point of connection to the 3′-oxygen; wherein Rrepresents a structure consisting of one or more atoms one of which iscovalently bound to the detectable label; and wherein Label representsthe detectable label.

In an embodiment, the t-butyldithiomethyl linker has the structure:

wherein α represents the point of connection to the 3′-oxygen; wherein nis an integer which may be 1, 2, 3, 4, or 5; and wherein R′ represents astructure covalently attached to the detectable label.

In an embodiment, the detectable label is a dye, a fluorophore, afluorescence energy transfer tag, a chemiluminescent compound, achromophore, a mass tag, an electrophore, a mononucleotide, anoligonucleotide, or a combination thereof. In a further embodiment, thedetectable label is a fluorophore. In a further embodiment, thefluorophore is BodipyFL, R6G, ROX, Cy5, or Alexa488.

In an embodiment, the nucleotide analog is3′-O-Alexa488-t-butyldithiomethyl-dCTP,3′-O-Cy5-t-butyldithiomethyl-dGTP, 3′-O-Rox-t-butyldithiomethyl-dATP,3′-O-RG6-t-butyldithiomethyl-dTTP,3′-O-Alexa488-PEG₄-t-butyldithiomethyl-dCTP,3′-O-RG6-PEG₄-t-butyldithiomethyl-dTTP,3′-O-Rox-PEG₄-t-butyldithiomethyl-dATP, or3′-O-Cy5-PEG₄-t-butyldithiomethyl-dGTP.

In an embodiment, the nucleotide analog has the structure:

In an embodiment, the invention comprises a composition comprising atleast two different nucleotide analogs, wherein each nucleotide analogconsists of a different base and a different detectable label from eachother nucleotide analog present in the composition.

The invention also provides for a method for determining the identity ofa nucleotide at a predetermined position in a nucleic acid of interest,comprising:

-   -   a) providing        -   1) the nucleic acid of interest,        -   2) a nucleic acid polymerase,        -   3) a primer capable of hybridizing to said nucleic acid            immediately 3′ of such predetermined position,        -   4) four different nucleotide analogs of claim 1, each of            which consists of one of adenine or an analog of adenine,            guanine or an analog of guanine, cytosine or an analog of            cytosine, thiamine or an analog of thiamine, and a unique            detectable label;    -   b) incorporating one of said nucleotide analogs onto the end of        said primer to form an extension strand;    -   c) detecting the unique detectable label of the incorporated        nucleotide analog so as to thereby identify the incorporated        nucleotide analog on the end of said extension strand; and    -   d) based on the identity of the incorporated nucleotide,        determining the identity of the nucleotide at the predetermined        position.

In an embodiment, the method further comprises treating the extensionstrand of step (b) so as to cleave the t-butyldithiomethyl linker boundto the 3′-oxygen of the sugar and so as to produce a 3′-OH on the sugarand for producing an extension, remove the label from the extensionstrand to which another nucleotide analog may be added.

In an embodiment, treatment further comprises contacting the extensionstrand with tris-(2-carboxyethyl)phosphine (TCEP) ortris(hydroxypropyl)phosphine (THP).

In an embodiment, each nucleotide analog is a nucleotide triphosphate, anucleotide tetraphosphate, a nucleotide pentaphosphate, or a nucleotidehexaphosphate. In an embodiment, the nucleotide analog comprises adeoxyribose. In an embodiment, the polymerase is a DNA polymerase andthe nucleic acid is DNA. In an embodiment, the polymerase is a reversetranscriptase and the nucleic acid is RNA. In an embodiment, thenucleotide analog comprises a ribose. In an embodiment, the polymeraseis a DNA-based RNA polymerase and the nucleic acid is DNA. In anembodiment, the polymerase is an RNA-based RNA polymerase and thenucleic acid is RNA.

In an embodiment, the t-Butyldithiomethyl linker has the structure:

wherein α represents the point of connection to the 3′-oxygen; wherein Rrepresents one or more atoms through which a covalent connection isestablished to the detectable label; and wherein Label is the detectablelabel.

In an embodiment, the t-Butyldithiomethyl linker has the structure:

wherein α represents the point of connection to the 3′-oxygen; wherein nis 1, 2, 3, 4, or 5; and wherein R′ represents one or more atoms throughwhich a covalent connection is established to the detectable label.

In an embodiment, the detectable label is selected from the groupconsisting of a dye, a fluorophore, a combinatorial fluorescence energytransfer tag, a chemiluminescent compound, a chromophore, a mass tag, anelectrophore, a mononucleotide, an oligonucleotide, or a combinationthereof. In a further embodiment, the detectable label is a fluorophore.In a further embodiment, the fluorophore is selected from the groupconsisting of BodipyFL, R6G, ROX, Cy5, and Alexa488.

In an embodiment, each nucleotide analog is selected from the groupconsisting of 3-O-Alexa488-t-Butyldithiomethyl-dCTP,3′-O-Cy5-t-Butyldithiomethyl-dGTP, 3′-O-Rox-t-Butyldithiomethyl-dATP,3′-O-RG6-t-Butyldithiomethyl-dTTP,3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP,3′-O-RG6-PEG₄-t-Butyldithiomethyl-dTTP,3′-O-Rox-PEG4-t-Butyldithiomethyl-dATP,3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP.

In an embodiment, the structure of each labeled nucleotide analog isselected from:

In an embodiment, the nucleic acid of interest is immobilized on a solidsupport.

In a further embodiment, the nucleic acid of interest is immobilized onthe solid support via an azido linkage, an alkynyl linkage, a1,3-dipolar cycloaddition linkage, or a biotin-streptavidin interaction.

In a further embodiment, the solid support is in the form of a chip, abead, a well, a capillary tube, or a slide. In a further embodiment, thesolid support comprises gold, quartz, silica, or a plastic. In a furtherembodiment, the solid support is porous.

In an embodiment, the invention comprises a method of sequencing anucleic acid of interest which comprises repeatedly determining theidentity of each nucleotide present in the nucleic acid of interest.

In a further embodiment, the invention comprises a method ofsimultaneously sequencing a plurality of different nucleic acids ofinterest which comprises simultaneously sequencing each such nucleicacid.

The invention also provides for a process for producing a3′-O-Bodipy-t-Butyldithiomethyl-dNTP, comprising:

-   -   a) reacting,        -   1) a 5′-O-tert-Butyldimethylsilyl-nucleoside,        -   2) acetic acid,        -   3) acetic anhydride, and        -   4) DMSO            under conditions permitting the production of a            3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside;    -   b) contacting the        3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside        produced in step a) with trimethylamine, molecular sieve,        sulfuryl chloride, potassium p-toluenethiosulfonate, and        2,2,2,-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide, under        conditions permitting the production of a product having the        structure:

wherein B is a nucleobase:

-   -   c) contacting the product produced in step b) with        tetrabutylammonium fluoride THF solution under conditions        permitting the production of a product having the structure:

-   -   -   wherein B is a nucleobase;

    -   d) contacting the product produced in step c) with        tetrabutylammonium pyrophosphate,        2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one, iodine solution        and ammonium hydroxide under conditions permitting the        production of a 3-O-NH₂-t-Butyldithiomethyl-dNTP;

    -   e) contacting the 3-O-NH₂-t-Butyldithiomethyl-dNTP of step d)        with Bodipy FL-NHS ester under conditions permitting the        production of the 3′-O-Bodipy-t-Butyldithiomethyl-dNTP.

In an embodiment, the 3′-O-Bodipy-t-Butyldithiomethyl-dNTP is a3′-O-Bodipy-t-Butyldithiomethyl-dATP or an analog thereof,3′-O-Bodipy-t-Butyldithiomethyl-dTTP or an analog thereof,3′-O-Bodipy-t-Butyldithiomethyl-dGTP or an analog thereof, or3′-O-Bodipy-t-Butyldithiomethyl-dCTP.

In a further embodiment, the 3′-O-Bodipy-t-Butyldithiomethyl-dNTP is3′-O-Bodipy-t-Butyldithiomethyl-dTTP.

The invention also provides for a process for producing a3′-O-Bodipy-PEG₄-t-Butyldithiomethyl-dNTP, comprising:

-   -   a) reacting,        -   1) a 5′-O-tert-Butyldimethylsilyl-nucleoside,        -   2) acetic acid.        -   3) acetic anhydride, and        -   4) DMSO            under conditions permitting the production of a            3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside;    -   b) contacting the        3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside        produced in part a) with trimethylamine, molecular sieve,        sulfuryl chloride, potassium p-toluenethiosulfonate, and        2,2,2,-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide, under        conditions permitting the production of a product having the        structure:

wherein B is a nucleobase:

-   -   c) contacting the product produced in step b) with        tetrabutylammonium fluoride THF solution under conditions        permitting the production of a product having the structure:

-   -   -   wherein B is a nucleobase;

    -   d) contacting the product produced in step c) with        tetrabutylammonium pyrophosphate,        2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one, iodine solution        and ammonium hydroxide under conditions permitting the        production of a 3-O-NH₂-t-Butyldithiomethyl-dNTP;

    -   e) contacting the 3-O-NH₂-t-Butyldithiomethyl-dNTP of step d)        with Bodipy-PEG₄-Acid, N,N-disuccinimidyl carbonate, and        4-dimethylaminopyridine under conditions permitting the        production of the 3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dNTP.

In an embodiment, the 3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dNTP is a3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dATP or an analog thereof,3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dTTP or an analog thereof,3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dGTP or an analog thereof, or3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dCTP.

In a further embodiment, the 3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dNTPis 3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dTTP.

The invention also provides for a process for producing a3′-O-Rox-t-Butyldithiomethyl-dATP, comprising:

-   -   a) reacting,        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine,            and        -   2) acetic acid, acetic anhydride and DMSO            under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine:    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of        3′-O-NH₂-t-Butyldithiomethyl-dATP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dATP produced in        step d) with ROX-NHS ester under conditions permitting the        production of the 3′-O-Rox-t-Butyldithiomethyl-dATP.

The invention also provides for a process for producing a3′-O-Rox-PEG₄-t-Butyldithiomethyl-dATP, comprising:

-   -   a) reacting,        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine,            and        -   2) acetic acid, acetic anhydride and DMSO under conditions            permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of        3′-O-NH₂-t-Butyldithiomethyl-dATP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dATP produced in        step d) with ROX-PEG₄-Acid, N,N-disuccinimidyl carbonate, and        4-dimethylaminopyridine under conditions permitting the        production of the 3′-O-Rox-PEG₄t-Butyldithiomethyl-dATP.

The invention also provides for a process for producing a3′-O-Alexa488-t-Butyldithiomethyl-dCTP, comprising:

-   -   a) reacting        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine,            and        -   2) acetic acid, acetic anhydride and DMSO            under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of a        3′-O-NH₂-t-Butyldithiomethyl-dCTP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dCTP produced in        step d) with Alexa488-NHS ester under conditions permitting the        production of the 3′-O-Alexa488-t-Butyldithiomethyl-dCTP.

The invention also provides for a process for producing a3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP, comprising:

-   -   a) reacting        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine,            and        -   2) acetic acid, acetic anhydride and DMSO under conditions            permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of a        3′-O-NH₂-t-Butyldithiomethyl-dCTP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dCTP produced in        step d) with Alexa488-PEG₄-NHS ester, N,N-disuccinimidyl        carbonate, and 4-dimethylaminopyridine under conditions        permitting the production of the        3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP.

The invention also provides for a process for producing a3′-O-Cy5-t-Butyldithiomethyl-dGTP, comprising:

-   -   a) reacting        -   1) a 2′-deoxyguanosine, and        -   2) tert-butyldimethylsilyl chloride, imidazole, and            N,N-dimethylformamide dimethyl acetal.            under conditions permitting the formation of a            N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine;    -   b) contacting the        N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine produced        in step a) with acetic acid acetic anhydride and DMSO under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with trimethylamine,        molecular sieves, sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   d) contacting the product of step c) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   e) contacting product of step d) with tetrabutylammonium        pyrophosphate, I-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of        3′-O-NH₂-t-Butyldithiomethyl-dGTP;    -   f) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dGTP produced in        step e) with Cy5-NHS under conditions permitting the production        of the 3′-O-Cy5-t-Butyldithiomethyl-dGTP.

The invention also provides for a process for producing a3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP, comprising:

-   -   a) reacting        -   1) a 2′-deoxyguanosine, and        -   2) tert-butyldimethylsilyl chloride, imidazole, and            N,N-dimethylformamide dimethyl acetal,            under conditions permitting the formation of a            N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine;    -   b) contacting the        N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine produced        in step a) with acetic acid acetic anhydride and DMSO under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with trimethylamine,        molecular sieves, sufurYl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   d) contacting the product of step c) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   e) contacting product of step d) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, iodine solution and ammonium hydroxide under        conditions permitting the production of        3′-O-NH₂-t-Butyldithiomethyl-dGTP;    -   f) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dGTP produced in        step e) with Cy5-PEG₄-NHS under conditions permitting the        production of the 3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP.

In certain embodiments of the invention, the label comprises a pluralityof identical Raman-scattering moieties. In other embodiments, the tagcomprises a plurality of different Raman-scattering moieties. In certainspecific embodiments, the tag comprises 3, 9, or 27 Raman-scatteringmoieties. In an embodiment, the plurality of Raman-scattering moietiesforms a linear tag. In another embodiment, the plurality ofRaman-scattering moieties forms a non-linear tag. In a preferredembodiment, the non-linear tag is a dendrimer tag. In an embodiment, thetag has a Raman spectroscopy peak with wavenumber from 2125 cm⁻¹ to 2260cm⁻¹.

In another embodiment the polymerase or polymerases are tethered to thenoble metal nanoparticles. In another embodiment the noble metalnanoparticles are silver and/or gold nanoparticles. In anotherembodiment the polymerase or polymerases have 1 or more attached and/orconjugated noble metal nanoparticles, wherein the noble metalnanoparticles are a surface-enhanced Raman spectroscopy (SERS)substrates. In another embodiment the noble metal nanoparticles areeither gold or silver nanoparticles. In another embodiment the metalnanoparticles of the polymerase or polymerases are between 3 nm and 10nm. In another embodiment the polymerase or polymerases have 2, 3, 4, or5 metal nanoparticles. In another embodiment the metal nanoparticles ofthe polymerase or polymerases are attached and/or conjugated to thepolymerase 1 nm-3 nm from the active site of the polymerase. In anotherembodiment the metal nanoparticles of the polymerase or polymerases areattached and/or conjugated to the polymerase or polymerases 1 nm-3 nmfrom the active site of the polymerase, thereby creating a region ofenhanced sensitivity for surface enhanced Raman spectroscopy (SERS) atthe active site. In another embodiment the metal nanoparticles areattached and/or conjugated to the polymerase such that when a nucleosideand/or nucleotide are in the active site of the polymerase, and whereinthe nucleoside and/or nucleotide are tagged with a Raman activemolecule, the metal nanoparticles are located 1 nm-3 nm from the Ramanactive molecule. In another embodiment the attached and/or conjugatedmetal nanoparticles of the polymerase create a region of enhancedsensitivity for surface enhanced Raman spectroscopy (SERS) at thelocation of the Raman active molecule.

The invention provides for a nucleotide analog consisting of (i) a base,(ii) a sugar, and (iii) a t-butyldithiomethyl linker bound to the3′-oxygen of the deoxyribose of the sugar.

Herein is further disclosed a method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first        type of nucleotide analogue under conditions permitting the        nucleotide polymerase to catalyze incorporation of the        nucleotide analogue into the primer if the nucleotide analogue        is complementary to a nucleotide residue of the single-stranded        DNA that is immediately 5′ to a nucleotide residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, so as to form a DNA extension product,        wherein the nucleotide analogue has the structure:

wherein base is any one of adenine, guanine, thymine, cytosine, uracil,or an analogue thereof, wherein Cleavable Moiety is a cleavablet-butyldithiomethyl moiety that when bound to the 3′-O prevents anucleotide polymerase from catalyzing a polymerase reaction with the3′-O of the nucleotide analogue, wherein Anchor is an anchor moiety thatis a small chemical moiety that orthogonally and rapidly reacts with acomplementary binding molecule thereby forming a conjugate of the anchormoiety and binding molecule, wherein ω represents a structure consistingof one or more atoms of which is covalently bound to both the cleavablet-butyldithiomethyl moiety and the anchor moiety, wherein the identityof the anchor is predetermined and is correlated to the identity of thebase,

-   -   b) contacting the single-stranded DNA of step a) with a binding        molecule complementary to the anchor of the nucleotide analogue        of step a), wherein the binding molecule has the structure:

-   -   -   wherein binder is a chemical group that orthogonally and            rapidly reacts with the anchor moiety, thereby forming a            conjugate of the binding molecule and the anchor moiety, and            Label is a detectable label,

    -   c) removing any nucleotide analogue not incorporated into the        primer in step a);

    -   d) detecting the presence of any detectable label so as to        thereby determine whether the nucleotide analogue of step a) was        incorporated so as to thereby determine the identity of the        complementary nucleotide residue in the single-stranded DNA, and        -   wherein if the base of the nucleotide analogue a) is not            complementary to the nucleotide residue of the            single-stranded DNA which is immediately 5′ to the            nucleotide residue of the single-stranded DNA hybridized to            the 3′ terminal nucleotide residue of the primer, then            iteratively repeating steps a) through c) with a second,            third, and then fourth type of nucleotide analogue, wherein            each different type of nucleotide analogue has a different            base from each other type of nucleotide analogue, until the            nucleotide analogue has a base that is complementary,

    -   e) cleaving the cleavable t-butyldithiomethyl moiety, so as to        thereby create a 3′-OH; and

    -   f) iteratively performing steps a) through e) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product, wherein each type of nucleotide analogue has the        structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein ω represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,        -   wherein the base of each type of nucleotide analogue is            independently different from the base of the remaining three            types of nucleotide analogue, wherein the anchor of each            type of nucleotide analogue is independently different from            the anchor of the remaining three types of nucleotide            analogue, wherein the anchor of each type of nucleotide            analogue orthogonally and rapidly reacts with a different            binding molecule from each of the remaining three types of            nucleotide analogue;

    -   b) contacting the single-stranded DNA of step a) with a first,        second, third, and fourth type of binding molecule, under        conditions permitting the anchor of the nucleotide analogue        incorporated in step a) to orthogonally and rapidly react with a        complementary binding molecule thereby binding the binding        molecule to the anchor,        -   wherein the first, second, third, and fourth type of binding            molecule each have the structure:

-   -   -   wherein binder is a small chemical group that orthogonally            and rapidly reacts with an anchor, and wherein Label is a            predetermined detectable label correlated to the identity of            the type of binding molecule, wherein the binder of each            type of binding molecule is different from the binder of the            remaining three types of binding molecule, wherein the first            type of binding molecule and the first type of nucleotide            analogue, the second type of binding molecule and second            type of nucleotide analogue, the third type of binding            molecule and third type of nucleotide analogue, and the            fourth type of binding molecule and the fourth type of            nucleotide analogue are respectively complementary and            thereby orthogonally and rapidly react thereby forming a            conjugate of each individual type of binding molecule an            individual type of nucleotide analogue;

    -   c) determining the identity of the detectable label of the        nucleotide analogue incorporated in step a) so as to thereby        determine the identity of the incorporated nucleotide analogue        and the identity of the complementary nucleotide residue in the        single-stranded DNA;

    -   d) cleaving the cleavable t-butyldithiomethyl moiety, so as to        thereby create a 3′-OH; and

    -   e) iteratively performing steps a) through d) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first and second types of nucleotide analogue            have the structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Linker is a            cleavable t-butyldithiomethyl moiety that when bound to the            3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein Label is a predetermined detectable label,            and wherein R represents a structure consisting of one or            more atoms of which is covalently bound to both the            cleavable t-butyldithiomethyl moiety and the detectable            label,        -   wherein the label of the first type of nucleotide analogue            is different form the label of the second type of nucleotide            analogue, wherein the base of each of the first and second            type of nucleotide analogue is independently different from            the base of each of the three remaining types of nucleotide            analogue,        -   wherein the third and fourth type of nucleotide analogue has            the structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein ω represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,        -   wherein the base of the third and fourth type of nucleotide            analogue is independently different from the base of each of            the three remaining types of nucleotide analogue, wherein            the anchor of the third type of nucleotide analogue is            different from the anchor of the fourth type of nucleotide            analogue:

    -   b) removing any nucleotide analogues not incorporated in step        a);

    -   c) detecting the presence of either the detectable label of the        first or second type of nucleotide analogue incorporated in        step a) so as to thereby determine the identity of the        incorporated nucleotide analogue and the identity of the        complementary nucleotide residue in the single-stranded DNA,        -   wherein if the base of the first and second type of            nucleotide is not complementary, contacting the            single-stranded DNA with a first and second type of binding            molecule, wherein the first and second type of binding            molecule have the structure:

-   -   -   wherein binder is a small chemical group that orthogonally            and rapidly reacts with an anchor, and wherein Label is a            predetermined detectable label correlated to the identity of            the binding molecule, wherein the detectable label of the            first type of binding molecule is the same as the detectable            label of the first type of nucleotide analogue, wherein the            detectable label of the second type of binding molecule is            the same as the detectable label of the second type of            nucleotide analogue, wherein the binder of the first type of            binding molecule orthogonally and rapidly reacts with the            anchor of the third type of nucleotide analogue, and wherein            the second type of binding molecule orthogonally and rapidly            reacts with the anchor of the fourth type of nucleotide            analogue, and        -   removing any unbound binding molecule, and detecting the            presence of either the first or second binding molecule so            as to thereby determine the identity of the nucleotide            analogue incorporated in step a) and the identity of the            complementary nucleotide residue in the single-stranded DNA;

    -   d) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   e) iteratively performing steps a) through d) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first, second, and third type of nucleotide            analogue have the structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein w represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,        -   wherein the base of the first, second, and third type of            nucleotide analogue is independently different from the base            of each of the three remaining types of nucleotide analogue,            wherein the first, second, and third type of nucleotide            analogue each independently have a different anchor from one            another,        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Cleavable Moiety is            a cleavable t-butyldithiomethyl moiety that when bound to            the 3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein the base of the fourth type of nucleotide            analogue is independently different from the base of each of            the three remaining types of nucleotide analogue:

    -   b) contact the single-stranded DNA of step a) with a first,        second, and third type of binding molecule, each type of binding        molecule having the structure:

-   -   -   wherein binder is a small chemical group correlated to the            identity of the type of binding molecule and that            orthogonally and rapidly reacts with an anchor, and wherein            Label is a detectable label,        -   wherein the binder of each type of binding molecule is            different from the binder of the remaining two types of            binding molecule, wherein the first type of binding molecule            and the first type of nucleotide analogue, the second type            of binding molecule and second type of nucleotide analogue,            and third type of binding molecule and third type of            nucleotide analogue are respectively complementary and            thereby orthogonally and rapidly react thereby binding each            individual type of binding molecule with an individual type            of nucleotide analogue:

    -   c) removing any nucleotide analogues from step a) not        incorporated into the primer;

    -   d) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   e) if a detectable label is detected in step d), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the first type of        nucleotide analogue;

    -   f) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the first type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   g) if a detectable label is detected in step f), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the second type of        nucleotide analogue;

    -   h) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   i) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   j) iteratively performing steps a) through i) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first and second types of nucleotide analogue            have the structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein ω represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,            -   wherein the base of the first and second type of                nucleotide analogue is independently different from the                base of each of the three remaining types of nucleotide                analogue, wherein the anchor of the first type of                nucleotide analogue is different from the anchor of the                second type of nucleotide analogue,        -   wherein the third type of nucleotide analogue has the            structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Linker is a            cleavable t-butyldithiomethyl moiety that when bound to the            3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein Label is a predetermined detectable label,            and wherein R represents a structure consisting of one or            more atoms of which is covalently bound to both the            cleavable t-butyldithiomethyl moiety and the detectable            label,        -   wherein the base of the third type of nucleotide analogue is            independently different from the base of each of the three            remaining types of nucleotide analogue,        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Cleavable Moiety is            a cleavable t-butyldithiomethyl moiety that when bound to            the 3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein the base of the fourth type of nucleotide            analogue is independently different from the base of each of            the three remaining types of nucleotide analogue;

    -   b) removing any nucleotide analogues from step a) not        incorporated into the primer;

    -   c) detecting whether there is a presence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the third type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   d) if an absence of detectable label bound to the incorporated        nucleotide of step a) is detected in step c), contacting the        single-stranded DNA with a first and second type of binding        molecule, wherein the first and second type of binding molecule        have the structure:

-   -   -   wherein binder is a small chemical group correlated to the            identity of the type of binding molecule and that            orthogonally and rapidly reacts with an anchor, and wherein            Label is a detectable label,        -   wherein the binder of each type of binding molecule is            different one another, wherein the first type of binding            molecule and the first type of nucleotide analogue, and the            second type of binding molecule and second type of            nucleotide analogue, respectively complementary and thereby            orthogonally and rapidly react thereby binding each            individual type of binding molecule with an individual type            of nucleotide analogue;

    -   e) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   f) if a detectable label is detected in step e), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the first type of        nucleotide analogue;

    -   g) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   h) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   i) iteratively performing steps a) through h) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first, second, and third type of nucleotide            analogue have the structure:

-   -   -   -   wherein base is any one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein                Cleavable Moiety is a cleavable t-butyldithiomethyl                moiety that when bound to the 3′-O prevents a nucleotide                polymerase from catalyzing a polymerase reaction with                the 3′-O of the nucleotide analogue, wherein Anchor is                an anchor moiety that is a small chemical moiety that                orthogonally and rapidly reacts with a complementary                binding molecule thereby forming a conjugate of the                anchor moiety and binding molecule, wherein ω represents                a structure consisting of one or more atoms of which is                covalently bound to both the cleavable                t-butyldithiomethyl moiety and the anchor moiety,

        -   wherein the base of the first, second, and third type of            nucleotide analogue is independently different from the base            of each of the three remaining types of nucleotide analogue,            wherein the first, second, and third type of nucleotide            analogue each independently have a different anchor from one            another,

        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   -   wherein base is one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein Linker                is a cleavable t-butyldithiomethyl moiety that when                bound to the 3′-O prevents a nucleotide polymerase from                catalyzing a polymerase reaction with the 3′-O of the                nucleotide analogue, wherein Label is a predetermined                detectable label, and wherein R represents a structure                consisting of one or more atoms of which is covalently                bound to both the cleavable t-butyldithiomethyl moiety                and the detectable label,

        -   wherein the base of the fourth type of nucleotide analogue            is independently different from the base of each of the            three remaining types of nucleotide analogue;

    -   b) removing all unincorporated nucleotide analogues from step        a);

    -   c) detecting whether there is a presence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   d) if an absence of detectable label bound to the incorporated        nucleotide of step a) is detected in step c), contacting the        single-stranded DNA with a first, second, and third type of        binding molecule, wherein the first, second, and third type of        binding molecule have the structure:

-   -   -   wherein binder is a small chemical group correlated to the            identity of the type of binding molecule and that            orthogonally and rapidly reacts with an anchor, and wherein            Label is a detectable label,        -   wherein the binder of each type of binding molecule is            different from one another, wherein the first type of            binding molecule and wherein the first type of binding            molecule and the first type of nucleotide analogue, the            second type of binding molecule and second type of            nucleotide analogue, and third type of binding molecule and            third type of nucleotide analogue are respectively            complementary and thereby orthogonally and rapidly react            thereby binding each individual type of binding molecule            with an individual type of nucleotide analogue;

    -   e) detecting whether there is a presence of detectable label        bound to the incorporated nucleotide of step a);

    -   f) contacting the single-stranded DNA with a means of cleaving        the detectable label and/or the binding molecule from the first        type of nucleotide analogue;

    -   g) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that the identity of the incorporated nucleotide is        of the first type of nucleotide analogue, and thereby the        identity of the complementary nucleotide residue in the        single-stranded DNA;

    -   h) if a detectable label is detected in step f), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the second type of        nucleotide analogue;

    -   i) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   j) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   k) iteratively performing steps a) through j) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first, second, and third types of nucleotide            analogue have the structure:

-   -   -   -   wherein base is one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein Linker                is a cleavable t-butyldithiomethyl moiety that when                bound to the 3′-O prevents a nucleotide polymerase from                catalyzing a polymerase reaction with the 3′-O of the                nucleotide analogue, wherein Label is a predetermined                detectable label, and wherein R represents a structure                consisting of one or more atoms of which is covalently                bound to both the cleavable t-butyldithiomethyl moiety                and the detectable label,

        -   wherein the base of the first, second, and third type of            nucleotide analogue is independently different from the base            of each of the three remaining types of nucleotide analogue,

        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   -   wherein base is one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein                Cleavable Moiety is a cleavable t-butyldithiomethyl                moiety that when bound to the 3′-O prevents a nucleotide                polymerase from catalyzing a polymerase reaction with                the 3′-O of the nucleotide analogue, wherein the base of                the fourth type of nucleotide analogue is independently                different from the base of each of the three remaining                types of nucleotide analogue:

    -   b) removing all unincorporated nucleotide analogues from step        a);

    -   c) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   d) if a detectable label is detected in step c), contacting the        single-stranded DNA with a means of cleaving the detectable        label from the first type of nucleotide analogue;

    -   e) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the first type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   f) if a detectable label is detected in step e), contacting the        single-stranded DNA with a means of cleaving the detectable        label from the second type of nucleotide analogue;

    -   g) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   h) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   i) iteratively performing steps a) through h) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

A further embodiment of the subject invention is a method fordetermining the identity of a nucleotide at a predetermined position ina nucleic acid of interest, comprising:

-   -   a) providing        -   1) the nucleic acid of interest,        -   2) a nucleic acid polymerase,        -   3) a primer capable of hybridizing to said nucleic acid            immediately 3′ of such predetermined position,        -   4) four different nucleotide analogues, wherein each            nucleotide analogue comprises (i) a base (ii) a sugar,            and (iii) a cleavable t-butyldithiomethyl moiety covalently            attached to a 3′-oxygen of the sugar, and            -   wherein the base of each analogue consists of one of                adenine or an analogue of adenine, guanine or an                analogue of guanine, cytosine or an analogue of                cytosine, thiamine or an analogue of thiamine, and a                unique detectable label;    -   b) incorporating one of said nucleotide analogues onto the end        of said primer to form an extension strand,    -   c) detecting the unique detectable label of the incorporated        nucleotide analogue so as to thereby identify the incorporated        nucleotide analogue on the end of said extension strand; and    -   d) based on the identity of the incorporated nucleotide,        determining the identity of the nucleotide at the predetermined        position.

3′-O Modified Nucleotides and Sequencing Methods:

Herein described are various approaches for DNA Sequencing by Synthesis(SBS) using 3′-O-reversibly-blocked nucleotide analogues. Thesenucleotide analogues include molecules with the following structures:3′-O-CleavableLinker-Label-dNTPs, 3′-O-CleavableLinker-Anchor-dNTPs and3′-O-CleavableGroup-dNTPs. The Cleavable Linker includes chemicallycleavable and photocleavable linkers. The “Anchor” refers to a smallchemical moiety that orthogonally and rapidly reacts with anotherchemical group that carries a detectable label. The Cleavable Grouprefers to a small chemical moiety that can be cleaved by either chemicalor photochemical means. Numerous schemes are provided to perform SBSusing the molecules comprising the three classes of nucleotide analogues(described above) in 1-color, 2-color, or 4-color formats.

Also herein disclosed are the design, synthesis, and use of novel 3′reversibly labeled nucleotides having various 3′-O-t-butyldithiomethyl(3′-O-DTM) modifications serving as the linkage to attach a reporter tothe nucleotides, thereby permitting the nucleotides to be “scarless”nucleotide reversible terminators (NRT) for DNA sequencing by synthesis(SBS). The 3′ attached reporter may be fluorescent. Such novel NRTs maybe employed in a set for use in SBS, wherein each NRT is 3′-O reversiblyblocked with a DTM group that is labeled with a fluorescent dye that hasa unique fluorescence emission corresponding to the type of base of eachnucleotide (e.g. a separate emission for A, T, G, and C respectively),thereby installing dual functions (serving as both a reversible blockerand a cleavable fluorescence reporter) to the 3′-O-modified nucleotideanalogues. During SBS, after a nucleotide is incorporated, and thefluorescent reporter imaged, the 3′-O-DTM-dye will be cleaved (cleavingagents may include THP or TCEP) to generate a 3′-OH group that is readyfor subsequent extension reactions. Many fluorescent dye species(several of which are identified herein) are suitable for polymeraseincorporation when attached to the 3′-O of these nucleotide analoguesvia DTM linkage.

Also described herein are, the three classes of nucleotide analoguespreviously mentioned (3′-O-CleavableLinker-Label-dNTPs,3′-O-CleavableLinker-Anchor-dNTPs and 3′-O-CleavableGroup-dNTPs),wherein the analogues are designed and synthesized based on thestructure of the nucleotide analogue3′-O-t-butyldithiomethyl-2′-deoxynucleoside-5′-triphosphates[3′-O-SS(DTM)-dNTPs]. More specifically, attachment of a fluorescent dyeto the DTM group at the 3′-end of the nucleotide analogue3′-O-SS(DTM)-dNTPs yields 3′-O-DTM-Dye-dNTPs; attachment of an “anchor”moiety to the DTM group at the 3′-end of the nucleotide analogue3′-O-SS(DTM)-dNTPs yields 3′-O-DTM-Anchor-dNTPs; when the cleavablegroup is the DTM itself at the 3′-end of the nucleotide analogue, thenucleotide analogue bears the parent structure itself[3′-O-SS(DTM)-dNTPs] without further Treatment of the DNA extensionproducts (described above) with tris (3-hydroxypropyl) phosphine (THP)in an aqueous buffer solution cleaves the DTM (SS) bond thereforeremoving the blocking group at the 3′-O position of the nucleotide,allowing the regeneration of a free OH group that is ready forsubsequent polymerase extension reactions to continually sequence DNA.DNA templates with homopolymer regions can be accurately sequenced usingthese nucleotide analogues.

Additionally, disclosed herein are the design, use, and synthesis ofnucleotide analogues that are attached with small “anchor” moieties tothe 3′-O position of the nucleotide analogues via a DTM linker. Sinceattaching smaller groups to the 3′-O position of the nucleotide analoguedoes not substantially interfere with the polymerase recognition ofthese molecules as substrates, these NRTs are more efficientlyincorporated to the growing DNA strand in SBS. After nucleotideincorporation, a corresponding labeled binding molecule tethered with afluorescent dye will orthogonally react with the anchor at the 3′-O endof the DNA extension product. Imaging of the fluorescent dye on this DNAextension product will identify the incorporated nucleotide for sequencedetermination. A general scheme to use these molecules for SBS is shownin FIG. 1.

The anchor moieties include a variety of orthogonally reactive oraffinitive functionalities with high efficiency and specificity, such asbiotin, azide, trans-cyclooctene (TCO) and phenyl boric acid (PBA),which will efficiently bind or react with streptavidin,dibenzocyclooctyne (DBCO) (John (2010); Shieha (2014)), tetrazine(TZ)(Marjoke (2013); Bergseid (2000)), and salicylhydroxamic acid (SHA)(Bergseid (2000)) respectively. The DNA polymerase will readilyincorporate these 3′-O-anchor-modified nucleotides to the growing DNAstrand to terminate DNA synthesis. Addition of the labeled bindingmolecules (such as different fluorophore-labeled streptavidin, DBCO, TZand SHA) to the corresponding primer extension product leads toorthogonal binding of the labeled binding molecules with thecorresponding “anchor” moiety in the 3′ end of the primer extensionproduct; after washing away the unbound labeled molecule, the detectionof the unique label attached to the 3′ end of the primer extensionproduct determines the identity of the incorporated nucleotide.

In addition to performing four-color SBS using the abovementionednucleotide analogues, these molecules also allow a wide spectrum of newDNA sequencing methods including one-color or two-color SBS at thesingle-molecule level or at an ensemble level. Instead of attaching asingle dye to the labeled binding molecules, multiple dyes can also beattached to the incorporated nucleotide through conjugation with thelabeled binding molecules that carry multiple-dyes (or dendrimerslabeled with multiple dyes), so that the amplification of fluorescentsignals can be achieved to facilitate single-molecule detection of theDNA extension product via SBS. Two-color SBS can be achieved byconnecting a binding molecule to a Fluorescence Resonance EnergyTransfer (FRET) cassette formed by two different fluorescent dyes, withdistinct emissions, which generate four different FRET signal signaturesto identify the four DNA bases (A, C, G, T) (Anthony (2001), Ju (1999)).If each labeled binding molecule is constructed by conjugation with adye reporter using a uniquely-cleavable linker for labeling the DNAextension product, different cleavage methods can be used for theselective removal of the dye from the DNA extension product; thedetected signal changes will therefore determine the incorporatednucleotide at the single-molecule level, or at the ensemble level, toperform SBS. A well-established cleavable linker toolbox [Azo (Leriche(2010), Budin (2010)), Dimethylketal (Bindaulda (2013)) Dde(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Ellis (2003)), ally andnitrobenzyl (Ju (2003), Li (2003), Ju (2006), Wu (2007))] is availableto develop the linkage between the labeled binding molecules and thereporting dye. These linkers can be readily cleaved under specificconditions by mild treatment with sodium dithionite (Na₂S₂O₄), weakacid, hydrazine (N₂H₄), Pd(0) and light-irradiation, respectively.

The invention provides for a nucleotide analogue comprised of (i) abase, (ii) a sugar, and (iii) a cleavable/-butyldithiomethyl moietybound to the 3′-oxygen of the deoxyribose of the sugar. In anembodiment, the sugar is a deoxyribose. In an embodiment, the sugar is aribose.

In an embodiment, the nucleotide analogue is a nucleotide monophosphate,a nucleotide diphosphate, a nucleotide triphosphate, a nucleotidetetraphosphate, a nucleotide pentaphosphate, or a nucleotidehexaphosphate.

In a further embodiment, the base of the analogue is adenine or ananalogue of adenine, guanine or an analogue of guanine, cytosine or ananalogue of cytosine, thymine or an analogue of thymine, or uracil or ananalogue of uracil.

In a further embodiment, the cleavable moiety may be cleaved by a watersoluble phosphine, thereby resulting in a 3′-OH. In a furtherembodiment, the water soluble phosphine istris-(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine(THP).

In a further embodiment, the cleavable t-butyldithiomethyl moiety hasthe structure:

wherein α represents the point of connection to the 3′-oxygen.

In a further embodiment the cleavable t-butyldithiomethyl moiety has thestructure:

wherein α represents the point of connection to the 3′-oxygen andwherein n is an integer which may be 1, 2, 3, 4, or 5.

In another embodiment the nucleotide analogue has the structure:

In another embodiment, the nucleotide analogue may further comprise adetectable label. In a further embodiment the cleavablet-butyldithiomethyl moiety has the structure:

wherein α represents the point of connection to the 3′-oxygen; wherein Rrepresents a structure consisting of one or more atoms one of which iscovalently bound to the detectable label; and wherein Label representsthe detectable label.

In a further embodiment the cleavable t-butyldithiomethyl moiety has thestructure:

wherein α represents the point of connection to the 3′-oxygen; wherein nis an integer which may be 1, 2, 3, 4, or 5; and wherein R′ representsone or more atoms through which a covalent connection is established tothe detectable label.

In a further embodiment, the nucleotide analogue has the structure:

wherein Cleavable Moiety is the cleavable t-butyldithiomethyl moiety,wherein Label represents the detectable label, and wherein R′ representsone or more atoms through which a covalent connection is established tothe detectable label.

In a further embodiment, the detectable label is one or more of a dye, afluorophore, a fluorescence energy transfer tag, a chemiluminescentcompound, a chromophore, a mass tag, an electrophore, a mononucleotide,an oligonucleotide, or a combination thereof.

In another embodiment, the the detectable label is a fluorophore. In yeta further embodiment, the fluorophore is BodipyFL, R6G, ROX, Cy5, orAlexa488.

In a further embodiment, the nucleotide analog is3′-O-Alexa488-t-butyldithiomethyl-dCTP,3′-O-Cy5-t-butyldithiomethyl-dGTP, 3′-O-Rox-t-butyldithiomethyl-dATP,3′-O-RG6-t-butyldithiomethyl-dTTP,3′-O-Alexa488-PEG₄-t-butyldithiomethyl-dCTP,3′-O-RG6-PEG₄-t-butyldithiomethyl-dTTP,3′-O-Rox-PEG₄-t-butyldithiomethyl-dATP, or3‘-O-Cy’5-PEG₄-t-butyldithiomethyl-dGTP.

In a further embodiment, the nucleotide analogue has the structure:

In yet another embodiment, the nucleotide analogue may further comprisean anchor, wherein the anchor is a predetermined small chemical moietycorrelated to the identity of the base and that orthogonally and rapidlyreacts with a complementary binding molecule thereby binding the anchorand binding molecule.

In a further embodiment, the nucleotide analogue has the structure:

wherein base is one of adenine, guanine, thymine, cytosine, uracil, oran analogue thereof, wherein Cleavable Moiety is the cleavablet-butyldithiomethyl moiety, wherein Anchor is the anchor moiety, andwherein ω represents a structure consisting of one or more atoms ofwhich is covalently bound to both the t-butyldithiomethyl cleavablemoiety and the anchor moiety.

In a further embodiment of the nucleotide analogue, the anchor has thestructure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety.

In yet a further embodiment, the anchor of the nucleotide analogueorthogonally and rapidly reacts with a complementary binding moleculethereby forming a conjugate of the anchor and binding molecule, whereinthe binding molecule has the structure:

wherein binder is a small chemical group correlated to the identity ofthe type of binding molecule and that orthogonally and rapidly reactswith an anchor, and wherein Label is a detectable label.

In a further embodiment, the detectable label of the complementarybinding molecule is selected from the group consisting of one or moredyes, fluorophores, combinatorial fluorescence energy transfer tags,chemiluminescent compounds, chromophores, mass tags, electrophores,mononucleotides, oligonucleotides, or combinations thereof.

In a further embodiment the detectable label of the complementarybinding molecule comprises one or more fluorescence energy transfertags. In a further embodiment the complementary binding molecule furthercomprises one or more FRET cassettes. In yet a further embodiment theFRET cassettes comprise one or more dSpacer monomers. In yet a furtherembodiment, the complementary binding molecule has the structure:

wherein T1 is a point of attachment for one or more fluorescent energydonor or acceptor, and T2 is a point of attachment for one or more ofthe complementary energy donor or acceptor to that in T1, wherein n isan integer between 1 and 20, and R represents the point of attachment tothe binder of the binding molecule.

In another embodiment, the detectable label of the complementary bindingmolecule is one or more fluorophore. In a further embodiment, thefluorophore of the detectable label of the complementary bindingmolecule is selected from the group consisting of BodipyFL. R6G, ROX,Cy5, and Alexa488.

In certain embodiments of the invention, the label comprises a pluralityof identical Raman-scattering moieties. In other embodiments, the tagcomprises a plurality of different Raman-scattering moieties. In certainspecific embodiments, the tag comprises 3, 9, or 27 Raman-scatteringmoieties. In an embodiment, the plurality of Raman-scattering moietiesforms a linear tag. In another embodiment, the plurality ofRaman-scattering moieties forms a non-linear tag. In a anotherembodiment, the non-linear tag is a dendrimer tag. In an embodiment, thetag has a Raman spectroscopy peak with wavenumber from 2125 cm⁻¹ to 2260cm⁻¹.

In another embodiment the nucleotide analogues are use in conjunctionwith a nucleotide polymerase or polymerases that are tethered to noblemetal nanoparticles. In another embodiment the noble metal nanoparticlesare silver and/or gold nanoparticles. In another embodiment thepolymerase or polymerases have 1 or more attached and/or conjugatednoble metal nanoparticles, wherein the noble metal nanoparticles are asurface-enhanced Raman spectroscopy (SERS) substrates. In anotherembodiment the noble metal nanoparticles are either gold or silvernanoparticles. In another embodiment the metal nanoparticles of thepolymerase or polymerases are between 3 nm and 10 nm. In anotherembodiment the polymerase or polymerases have 2, 3, 4, or 5 metalnanoparticles. In another embodiment the metal nanoparticles of thepolymerase or polymerases are attached and/or conjugated to thepolymerase 1 nm-3 nm from the active site of the polymerase. In anotherembodiment the metal nanoparticles of the polymerase or polymerases areattached and/or conjugated to the polymerase or polymerases 1 nm-3 nmfrom the active site of the polymerase, thereby creating a region ofenhanced sensitivity for surface enhanced Raman spectroscopy (SERS) atthe active site. In another embodiment the metal nanoparticles areattached and/or conjugated to the polymerase such that when a nucleosideand/or nucleotide are in the active site of the polymerase, and whereinthe nucleoside and/or nucleotide are tagged with a Raman activemolecule, the metal nanoparticles are located 1 nm-3 nm from the Ramanactive molecule. In another embodiment the attached and/or conjugatedmetal nanoparticles of the polymerase create a region of enhancedsensitivity for surface enhanced Raman spectroscopy (SERS) at thelocation of the Raman active molecule.

In a further embodiment, the binder of the complementary bindingmolecule comprises:

-   -   a) a compound comprising streptavidin having the structure:

or

-   -   b) a compound comprising the structure:

wherein α represents one or more atoms through which a covalentconnection is established to the detectable label.

In a further embodiment, if the anchor of the nucleotide analogue hasthe structure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butldithiomethyl moiety,then the anchor can orthogonally and rapidly react with a binder of acomplimentary binding molecule, wherein said binder comprisesstreptavidin, and has the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label, thereby forming a conjugate havingthe structure:

wherein α is one or more atoms through which a covalent connection isestablished to detectable label, and wherein ω is one or more atomsthrough which a covalent connection is established to the cleavablet-butyldithiomethyl moiety.

In a further embodiment, the nucleotide analogue has the structure:

wherein base is one of adenine, guanine, thymine, cytosine, uracil, oran analogue thereof.

In a further embodiment the complementary binding molecule to thenucleotide analogue comprises streptavidin, and wherein thecomplementary binding molecule has the structure:

In another embodiment, if the anchor of the nucleotide analogue has thestructure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety,then the anchor can orthogonally and rapidly react with a binder of acomplimentary binding molecule, wherein said binder has the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label, thereby forming a conjugate havingthe structure:

wherein α is one or more atoms through which a covalent connection isestablished to detectable label, and wherein ω is one or more atomsthrough which a covalent connection is established to the cleavablet-butyldithiomethyl moiety.

In a further embodiment, the nucleotide analogue has the structure:

wherein base is one of adenine, guanine, thymine, cytosine, uracil, oran analogue thereof.

In a further embodiment, the complementary binding molecule to thenucleotide analogue has the structure:

In another embodiment, wherein if the anchor has the structure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety,then the anchor orthogonally and rapidly reacts with a binder of acomplimentary binding molecule, wherein said binder has the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label, thereby forming a conjugate havingthe structure:

wherein α is one or more atoms through which a covalent connection isestablished to detectable label, and wherein ω is one or more atomsthrough which a covalent connection is established to the cleavablet-butyldithiomethyl moiety.

In further embodiment of the nucleotide analogue, the nucleotideanalogue has the structure:

wherein base is one of adenine, guanine, thymine, cytosine, uracil, oran analogue thereof.

In further embodiment the complementary binding to the nucleotideanalogue has the structure:

In a further embodiment, if the anchor has the structure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety,then the anchor can orthogonally and rapidly react with a binder of acomplimentary binding molecule, wherein said binder has the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label, thereby forming a conjugate havingthe structure:

wherein α is one or more atoms through which a covalent connection isestablished to detectable label, and wherein ω is one or more atomsthrough which a covalent connection is established to the cleavablet-butldithiomethyl moiety.

In further embodiment, the nucleotide analogue has the structure:

wherein base is one of adenine, guanine, thymine, cytosine, uracil, oran analogue thereof.

In further embodiment, the complementary binding molecule to thenucleotide analogue has the structure:

Herein is further disclosed a method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first        type of nucleotide analogue under conditions permitting the        nucleotide polymerase to catalyze incorporation of the        nucleotide analogue into the primer if the nucleotide analogue        is complementary to a nucleotide residue of the single-stranded        DNA that is immediately 5′ to a nucleotide residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, so as to form a DNA extension product,        wherein the nucleotide analogue has the structure:

wherein base is any one of adenine, guanine, thymine, cytosine, uracil,or an analogue thereof, wherein Cleavable Moiety is a cleavablet-butyldithiomethyl moiety that when bound to the 3′-O prevents anucleotide polymerase from catalyzing a polymerase reaction with the3′-O of the nucleotide analogue, wherein Anchor is an anchor moiety thatis a small chemical moiety that orthogonally and rapidly reacts with acomplementary binding molecule thereby forming a conjugate of the anchormoiety and binding molecule, wherein ω represents a structure consistingof one or more atoms of which is covalently bound to both the cleavablet-butyldithiomethyl moiety and the anchor moiety, wherein the identityof the anchor is predetermined and is correlated to the identity of thebase,

-   -   b) contacting the single-stranded DNA of step a) with a binding        molecule complementary to the anchor of the nucleotide analogue        of step a), wherein the binding molecule has the structure:

wherein binder is a chemical group that orthogonally and rapidly reactswith the anchor moiety, thereby forming a conjugate of the bindingmolecule and the anchor moiety, and Label is a detectable label,

-   -   c) removing any nucleotide analogue not incorporated into the        primer in step a);    -   d) detecting the presence of any detectable label so as to        thereby determine whether the nucleotide analogue of step a) was        incorporated so as to thereby determine the identity of the        complementary nucleotide residue in the single-stranded DNA, and        wherein if the base of the nucleotide analogue a) is not        complementary to the nucleotide residue of the single-stranded        DNA which is immediately 5′ to the nucleotide residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, then iteratively repeating steps a)        through c) with a second, third, and then fourth type of        nucleotide analogue, wherein each different type of nucleotide        analogue has a different base from each other type of nucleotide        analogue, until the nucleotide analogue has a base that is        complementary,    -   e) cleaving the cleavable t-butyldithiomethyl moiety, so as to        thereby create a 3′-OH; and    -   f) iteratively performing steps a) through e) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

In a further embodiment of the method, steps b) and c) are performedsimultaneously, or in the order step b) then step c) or in the orderstep c) then step b).

In a further embodiment of the method, different nucleotide analogueshave different anchors, and each different anchor is complementary to adifferent binding molecule.

In a further embodiment of the method the different binding moleculeseach have a different detectable label.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product, wherein each type of nucleotide analogue has the        structure:

wherein base is any one of adenine, guanine, thymine, cytosine, uracil,or an analogue thereof, wherein Cleavable Moiety is a cleavablet-butyldithiomethyl moiety that when bound to the 3′-O prevents anucleotide polymerase from catalyzing a polymerase reaction with the3′-O of the nucleotide analogue, wherein Anchor is an anchor moiety thatis a small chemical moiety that orthogonally and rapidly reacts with acomplementary binding molecule thereby forming a conjugate of the anchormoiety and binding molecule, wherein ω represents a structure consistingof one or more atoms of which is covalently bound to both the cleavablet-butyldithiomethyl moiety and the anchor moiety, wherein the base ofeach type of nucleotide analogue is independently different from thebase of the remaining three types of nucleotide analogue, wherein theanchor of each type of nucleotide analogue is independently differentfrom the anchor of the remaining three types of nucleotide analogue,wherein the anchor of each type of nucleotide analogue orthogonally andrapidly reacts with a different binding molecule from each of theremaining three types of nucleotide analogue;

-   -   b) contacting the single-stranded DNA of step a) with a first,        second, third, and fourth type of binding molecule, under        conditions permitting the anchor of the nucleotide analogue        incorporated in step a) to orthogonally and rapidly react with a        complementary binding molecule thereby binding the binding        molecule to the anchor, wherein the first, second, third, and        fourth type of binding molecule each have the structure:

wherein binder is a small chemical group that orthogonally and rapidlyreacts with an anchor, and wherein Label is a predetermined detectablelabel correlated to the identity of the type of binding molecule,wherein the binder of each type of binding molecule is different fromthe binder of the remaining three types of binding molecule, wherein thefirst type of binding molecule and the first type of nucleotideanalogue, the second type of binding molecule and second type ofnucleotide analogue, the third type of binding molecule and third typeof nucleotide analogue, and the fourth type of binding molecule and thefourth type of nucleotide analogue are respectively complementary andthereby orthogonally and rapidly react thereby forming a conjugate ofeach individual type of binding molecule an an individual type ofnucleotide analogue;

-   -   c) determining the identity of the detectable label of the        nucleotide analogue incorporated in step a) so as to thereby        determine the identity of the incorporated nucleotide analogue        and the identity of the complementary nucleotide residue in the        single-stranded DNA;    -   d) cleaving the cleavable t-butyldithiomethyl moiety, so as to        thereby create a 3′-OH; and    -   e) iteratively performing steps a) through d) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first and second types of nucleotide analogue            have the structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Linker is a            cleavable t-butyldithiomethyl moiety that when bound to the            3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein Label is a predetermined detectable label,            and wherein R represents a structure consisting of one or            more atoms of which is covalently bound to both the            cleavable t-butyldithiomethyl moiety and the detectable            label,        -   wherein the label of the first type of nucleotide analogue            is different form the label of the second type of nucleotide            analogue, wherein the base of each of the first and second            type of nucleotide analogue is independently different from            the base of each of the three remaining types of nucleotide            analogue,        -   wherein the third and fourth type of nucleotide analogue has            the structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein w represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,        -   wherein the base of the third and fourth type of nucleotide            analogue is independently different from the base of each of            the three remaining types of nucleotide analogue, wherein            the anchor of the third type of nucleotide analogue is            different from the anchor of the fourth type of nucleotide            analogue;

    -   b) removing any nucleotide analogues not incorporated in step        a);

    -   c) detecting the presence of either the detectable label of the        first or second type of nucleotide analogue incorporated in        step a) so as to thereby determine the identity of the        incorporated nucleotide analogue and the identity of the        complementary nucleotide residue in the single-stranded DNA,        -   wherein if the base of the first and second type of            nucleotide is not complementary, contacting the            single-stranded DNA with a first and second type of binding            molecule, wherein the first and second type of binding            molecule have the structure:

-   -   -   wherein binder is a small chemical group that orthogonally            and rapidly reacts with an anchor, and wherein Label is a            predetermined detectable label correlated to the identity of            the binding molecule, wherein the detectable label of the            first type of binding molecule is the same as the detectable            label of the first type of nucleotide analogue, wherein the            detectable label of the second type of binding molecule is            the same as the detectable label of the second type of            nucleotide analogue, wherein the binder of the first type of            binding molecule orthogonally and rapidly reacts with the            anchor of the third type of nucleotide analogue, and wherein            the second type of binding molecule orthogonally and rapidly            reacts with the anchor of the fourth type of nucleotide            analogue,        -   removing any unbound binding molecule, and detecting the            presence of either the first or second binding molecule so            as to thereby determine the identity of the nucleotide            analogue incorporated in step a) and the identity of the            complementary nucleotide residue in the single-stranded DNA;

    -   d) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   e) iteratively performing steps a) through d) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first, second, and third type of nucleotide            analogue have the structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein ω represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,        -   wherein the base of the first, second, and third type of            nucleotide analogue is independently different from the base            of each of the three remaining types of nucleotide analogue,            wherein the first, second, and third type of nucleotide            analogue each independently have a different anchor from one            another,        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Cleavable Moiety is            a cleavable t-butyldithiomethyl moiety that when bound to            the 3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein the base of the fourth type of nucleotide            analogue is independently different from the base of each of            the three remaining types of nucleotide analogue:

    -   b) contact the single-stranded DNA of step a) with a first,        second, and third type of binding molecule, each type of binding        molecule having the structure:

-   -   -   wherein binder is a small chemical group correlated to the            identity of the type of binding molecule and that            orthogonally and rapidly reacts with an anchor, and wherein            Label is a detectable label,        -   wherein the binder of each type of binding molecule is            different from the binder of the remaining two types of            binding molecule, wherein the first type of binding molecule            and the first type of nucleotide analogue, the second type            of binding molecule and second type of nucleotide analogue,            and third type of binding molecule and third type of            nucleotide analogue are respectively complementary and            thereby orthogonally and rapidly react thereby binding each            individual type of binding molecule with an individual type            of nucleotide analogue:

    -   c) removing any nucleotide analogues from step a) not        incorporated into the primer;

    -   d) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   e) if a detectable label is detected in step d), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the first type of        nucleotide analogue;

    -   f) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the first type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   g) if a detectable label is detected in step f), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the second type of        nucleotide analogue,

    -   h) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   i) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   j) iteratively performing steps a) through i) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first and second types of nucleotide analogue            have the structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein ω represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,        -   wherein the base of the first and second type of nucleotide            analogue is independently different from the base of each of            the three remaining types of nucleotide analogue, wherein            the anchor of the first type of nucleotide analogue is            different from the anchor of the second type of nucleotide            analogue,        -   wherein the third type of nucleotide analogue has the            structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Linker is a            cleavable t-butyldithiomethyl moiety that when bound to the            3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein Label is a predetermined detectable label,            and wherein R represents a structure consisting of one or            more atoms of which is covalently bound to both the            cleavable t-butyldithiomethyl moiety and the detectable            label,        -   wherein the base of the third type of nucleotide analogue is            independently different from the base of each of the three            remaining types of nucleotide analogue, wherein the fourth            type of nucleotide analogue has the structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Cleavable Moiety is            a cleavable t-butyldithiomethyl moiety that when bound to            the 3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein the base of the fourth type of nucleotide            analogue is independently different from the base of each of            the three remaining types of nucleotide analogue:

    -   b) removing any nucleotide analogues from step a) not        incorporated into the primer;

    -   c) detecting whether there is a presence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the third type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   d) if an absence of detectable label bound to the incorporated        nucleotide of step a) is detected in step c), contacting the        single-stranded DNA with a first and second type of binding        molecule, wherein the first and second type of binding molecule        have the structure:

-   -   -   wherein binder is a small chemical group correlated to the            identity of the type of binding molecule and that            orthogonally and rapidly reacts with an anchor, and wherein            Label is a detectable label,        -   wherein the binder of each type of binding molecule is            different one another, wherein the first type of binding            molecule and the first type of nucleotide analogue, and the            second type of binding molecule and second type of            nucleotide analogue, respectively complementary and thereby            orthogonally and rapidly react thereby binding each            individual type of binding molecule with an individual type            of nucleotide analogue;

    -   e) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   f) if a detectable label is detected in step e), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the first type of        nucleotide analogue;

    -   g) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   h) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   i) iteratively performing steps a) through h) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first, second, and third type of nucleotide            analogue have the structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein ω represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety.        -   wherein the base of the first, second, and third type of            nucleotide analogue is independently different from the base            of each of the three remaining types of nucleotide analogue,            wherein the first, second, and third type of nucleotide            analogue each independently have a different anchor from one            another,        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Linker is a            cleavable t-butyldithiomethyl moiety that when bound to the            3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein Label is a predetermined detectable label,            and wherein R represents a structure consisting of one or            more atoms of which is covalently bound to both the            cleavable t-butyldithiomethyl moiety and the detectable            label,        -   wherein the base of the fourth type of nucleotide analogue            is independently different from the base of each of the            three remaining types of nucleotide analogue;

    -   b) removing all unincorporated nucleotide analogues from step        a);

    -   c) detecting whether there is a presence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   d) if an absence of detectable label bound to the incorporated        nucleotide of step a) is detected in step c), contacting the        single-stranded DNA with a first, second, and third type of        binding molecule, wherein the first, second, and third type of        binding molecule have the structure:

-   -   -   wherein binder is a small chemical group correlated to the            identity of the type of binding molecule and that            orthogonally and rapidly reacts with an anchor, and wherein            Label is a detectable label,        -   wherein the binder of each type of binding molecule is            different from one another, wherein the first type of            binding molecule and wherein the first type of binding            molecule and the first type of nucleotide analogue, the            second type of binding molecule and second type of            nucleotide analogue, and third type of binding molecule and            third type of nucleotide analogue are respectively            complementary and thereby orthogonally and rapidly react            thereby binding each individual type of binding molecule            with an individual type of nucleotide analogue;

    -   e) detecting whether there is a presence of detectable label        bound to the incorporated nucleotide of step a);

    -   f) contacting the single-stranded DNA with a means of cleaving        the detectable label and/or the binding molecule from the first        type of nucleotide analogue;

    -   g) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that the identity of the incorporated nucleotide is        of the first type of nucleotide analogue, and thereby the        identity of the complementary nucleotide residue in the        single-stranded DNA;

    -   h) if a detectable label is detected in step f), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the second type of        nucleotide analogue;

    -   i) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   j) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   k) iteratively performing steps a) through j) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Herein is disclosed a further method for determining the nucleotidesequence of a single-stranded DNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first, second, and third types of nucleotide            analogue have the structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Linker is a            cleavable t-butyldithiomethyl moiety that when bound to the            3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein Label is a predetermined detectable label,            and wherein R represents a structure consisting of one or            more atoms of which is covalently bound to both the            cleavable t-butyldithiomethyl moiety and the detectable            label,        -   wherein the base of the first, second, and third type of            nucleotide analogue is independently different from the base            of each of the three remaining types of nucleotide analogue,        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Cleavable Moiety is            a cleavable t-butyldithiomethyl moiety that when bound to            the 3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein the base of the fourth type of nucleotide            analogue is independently different from the base of each of            the three remaining types of nucleotide analogue;

    -   b) removing all unincorporated nucleotide analogues from step        a);

    -   c) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   d) if a detectable label is detected in step c), contacting the        single-stranded DNA with a means of cleaving the detectable        label from the first type of nucleotide analogue;

    -   e) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the first type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   f) if a detectable label is detected in step e), contacting the        single-stranded DNA with a means of cleaving the detectable        label from the second type of nucleotide analogue;

    -   g) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   h) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   i) iteratively performing steps a) through h) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

In a further embodiment of the foregoing methods, the anchor of eachtype of nucleotide analogue having an anchor that forms a conjugate witha complementary binding molecule, each individually has the structure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety.

In a further embodiment, the detectable label of the complementarybinding molecule is selected from the group consisting of one or moredyes, fluorophores, combinatorial fluorescence energy transfer tags,chemiluminescent compounds, chromophores, mass tags, electrophores,mononucleotides, oligonucleotides, or combinations thereof.

In a further embodiment the detectable label of the complementarybinding molecule comprises one or more fluorescence energy transfertags.

In a further embodiment the complementary binding molecule furthercomprises one or more FRET cassettes. In a further embodiment the FRETcassettes comprise one or more dSpacer monomers.

In a further embodiment the complementary binding molecule has thestructure:

wherein T1 is a point of attachment for one or more fluorescent energydonor or acceptor, and T2 is a point of attachment for one or more ofthe complementary energy donor or acceptor to that in Ti, wherein n isan integer between 1 and 20, and R represents the point of attachment tothe binder of the binding molecule.

In a further embodiment the detectable label of the complementarybinding molecule is one or more fluorophore. In a further embodiment thefluorophore of the detectable label of the complementary bindingmolecule is selected from the group consisting of BodipyFL, R6G, ROX,Cy5, and Alexa488.

In certain embodiments of the invention, the label comprises a pluralityof identical Raman-scattering moieties. In other embodiments, the tagcomprises a plurality of different Raman-scattering moieties. In certainspecific embodiments, the tag comprises 3, 9, or 27 Raman-scatteringmoieties. In an embodiment, the plurality of Raman-scattering moietiesforms a linear tag. In another embodiment, the plurality ofRaman-scattering moieties forms a non-linear tag. In a anotherembodiment, the non-linear tag is a dendrimer tag. In an embodiment, thetag has a Raman spectroscopy peak with wavenumber from 2125 cm⁻¹ to 2260cm⁻¹.

In another embodiment the polymerase or polymerases are tethered to thenoble metal nanoparticles. In another embodiment the noble metalnanoparticles are silver and/or gold nanoparticles. In anotherembodiment the polymerase or polymerases have 1 or more attached and/orconjugated noble metal nanoparticles, wherein the noble metalnanoparticles are a surface-enhanced Raman spectroscopy (SERS)substrates. In another embodiment the noble metal nanoparticles areeither gold or silver nanoparticles. In another embodiment the metalnanoparticles of the polymerase or polymerases are between 3 nm and 10nm. In another embodiment the polymerase or polymerases have 2, 3, 4, or5 metal nanoparticles. In another embodiment the metal nanoparticles ofthe polymerase or polymerases are attached and/or conjugated to thepolymerase 1 nm-3 nm from the active site of the polymerase. In anotherembodiment the metal nanoparticles of the polymerase or polymerases areattached and/or conjugated to the polymerase or polymerases 1 nm-3 nmfrom the active site of the polymerase, thereby creating a region ofenhanced sensitivity for surface enhanced Raman spectroscopy (SERS) atthe active site. In another embodiment the metal nanoparticles areattached and/or conjugated to the polymerase such that when a nucleosideand/or nucleotide are in the active site of the polymerase, and whereinthe nucleoside and/or nucleotide are tagged with a Raman activemolecule, the metal nanoparticles are located 1 nm-3 nm from the Ramanactive molecule. In another embodiment the attached and/or conjugatedmetal nanoparticles of the polymerase create a region of enhancedsensitivity for surface enhanced Raman spectroscopy (SERS) at thelocation of the Raman active molecule.

In some embodiments of the invention, vibrational spectroscopy is usedto detect the presence of incorporated nucleotide analogs. Vibrationalspectroscopy is a spectrographic analysis where the sample isilluminated with incident radiation in order to excite molecularvibrations. Vibrational excitation, caused by molecules of the sampleabsorbing, reflecting or scattering a particular discrete amount ofenergy, is detected and can be measured. The two major types ofvibrational spectroscopy are infrared (usually FTIR) and Raman. If FTIRis employed, then the IR spectra of the nucleotide analogs are measured.If Raman is employed, then the Raman spectra of the nucleotide analogsis measured (for example of the nucleotide analogs and in the methodsdescribed herein).

Because of well-understood base-pairing rules, determining thewavenumber of the Raman spectroscopy peak of a dNTP analog incorporatedinto a primer or DNA extension product, and thereby the identity of thedNTP analog that was incorporated, permits identification of thecomplementary nucleotide residue in the single-stranded polynucleotidethat the primer or DNA extension product is hybridized to. Thus, if thedNTP analog that was incorporated has a unique wavenumber in the Ramanspectroscopy peak identifying it as comprising an adenine, a thymine, acytosine, or a guanine, then the complementary nucleotide residue in thesingle-stranded polynucleotide is identified as a thymine, an adenine, aguanine or a cytosine, respectively. The purine adenine (A) pairs withthe pyrimidine thymine (T). The pyrimidine cytosine (C) pairs with thepurine guanine (G). Similarly, with regard to RNA, if the dNTP analogthat was incorporated comprises an adenine, a uracil, a cytosine, or aguanine, then the complementary nucleotide residue in thesingle-stranded RNA is identified as a uracil, an adenine, a guanine ora cytosine, respectively.

In a further embodiment the binder of the complementary binding moleculeof each type of nucleotide analogue having an anchor comprises:

-   -   a) a compound comprising streptavidin having the structure:

orb) a compound comprising the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label.

In a further embodiment, if the anchor of a type of nucleotide analoguehas the structure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety,then the anchor orthogonally and rapidly reacts with a binder of acomplimentary binding molecule, wherein said binder comprisesstreptavidin, and has the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label, thereby forming a conjugate havingthe structure:

wherein α is one or more atoms through which a covalent connection isestablished to detectable label, and wherein ω is one or more atomsthrough which a covalent connection is established to the cleavablet-butyldithiomethyl moiety.

In a further embodiment the label is cleaved from the conjugatecomprising the type of nucleotide analogue and the binding molecule withcitric acid/Na₂HPO₄.

In a further embodiment the type of nucleotide analogue has thestructure:

wherein base is one of adenine, guanine, thymine, cytosine, uracil, orderivatives thereof.

In a further embodiment the complementary binding molecule comprisesstreptavidin, and wherein the complementary binding molecule has thestructure:

In a further embodiment the anchor of a type of nucleotide analogue hasthe structure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety,then the anchor orthogonally and rapidly reacts with a binder of acomplimentary binding molecule, wherein said binder has the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label, thereby forming a conjugate havingthe structure:

wherein α is one or more atoms through which a covalent connection isestablished to detectable label, and wherein ω is one or more atomsthrough which a covalent connection is established to the cleavablet-butyldithiomethyl moiety.

In a further embodiment the label is cleaved from the conjugatecomprising the type of nucleotide analogue and binding molecule withNa₂S₂O₄/H₂O.

In a further embodiment the type of nucleotide analogue has thestructure:

wherein base is one of adenine, guanine, thymine, cytosine, uracil, orderivatives thereof.

In a further embodiment, the complementary binding molecule has thestructure:

In a further embodiment, if the anchor of a type of nucleotide analoguehas the structure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety,then the anchor orthogonally and rapidly reacts with a binder of acomplimentary binding molecule, wherein said binder has the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label, thereby forming a conjugate havingthe structure:

wherein α is one or more atoms through which a covalent connection isestablished to detectable label, and wherein ω is one or more atomsthrough which a covalent connection is established to the cleavablet-butyldithiomethyl moiety.

In a further embodiment the type of nucleotide analogue has thestructure:

wherein base is one of adenine, guanine, thymine, cytosine, uracil, orderivatives thereof.

In a further embodiment the complementary binding molecule has thestructure:

In a further embodiment if the anchor of a type of nucleotide analoguehas the structure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety,then the anchor orthogonally and rapidly reacts with a binder of acomplimentary binding molecule, wherein said binder has the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label, thereby forming a conjugate havingthe structure:

wherein α is one or more atoms through which a covalent connection isestablished to detectable label, and wherein ω is one or more atomsthrough which a covalent connection is established to the cleavablet-butyldithiomethyl moiety.

In a further embodiment, the label is cleaved from the conjugatecomprising the type of nucleotide analogue and binding molecule withcitric acid/Na₂HPO₄.

In a further embodiment the type of nucleotide analogue has thestructure:

wherein base is one of adenine, guanine, thymine, cytosine, uracil, orderivatives thereof.

In a further embodiment, the complementary binding molecule has thestructure:

In a further embodiment the cleavable t-butyldithiomethyl moiety of eachtype of nucleotide analogue is a t-butyldithiomethyl linker, which hasthe structure:

wherein α represents the point of connection to the 3′-oxygen.

In a further embodiment the cleavable t-butyldithiomethyl linker has thestructure:

wherein α represents the point of connection to the 3′-oxygen; andwherein n is an integer which may be 1, 2, 3, 4, or 5.

In a further embodiment the cleavable t-butyldithiomethyl moiety may becleaved by a water soluble phosphine, thereby resulting in a 3′-OH. In afurther embodiment the water soluble phosphine istris-(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine(THP), wherein the cleavable t-butyldithiomethyl moiety may be cleavedby a water soluble phosphine, thereby resulting in a 3′-OH.

In further embodiments of the foregoing methods, the nucleotideanalogues having the structure:

may be nucleotide analogues having the structure:

A further embodiment of the subject invention is a method fordetermining the identity of a nucleotide at a predetermined position ina nucleic acid of interest, comprising:

-   -   a) providing        -   1) the nucleic acid of interest,        -   2) a nucleic acid polymerase,        -   3) a primer capable of hybridizing to said nucleic acid            immediately 3′ of such predetermined position,        -   4) four different nucleotide analogues, wherein each            nucleotide analogue comprises (i) a base (ii) a sugar,            and (iii) a cleavable t-butyldithiomethyl moiety covalently            attached to a 3′-oxygen of the sugar, and            -   wherein the base of each analogue consists of one of                adenine or an analogue of adenine, guanine or an                analogue of guanine, cytosine or an analogue of                cytosine, thiamine or an analogue of thiamine, and a                unique detectable label;    -   b) incorporating one of said nucleotide analogues onto the end        of said primer to form an extension strand;    -   c) detecting the unique detectable label of the incorporated        nucleotide analogue so as to thereby identify the incorporated        nucleotide analogue on the end of said extension strand; and    -   d) based on the identity of the incorporated nucleotide,        determining the identity of the nucleotide at the predetermined        position.

In a further embodiment, the extension strand of step (b) is treated soas to cleave the t-butyldithiomethyl moiety bound to the 3′-oxygen ofthe sugar and so as to produce a 3′-OH on the sugar and for producing anextension, remove the label from the extension strand to which anothernucleotide analogue may be added. In a further method, the treatmentcomprises contacting the extension strand withtris-(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine(THP).

In a further embodiment the t-Butyldithiomethyl linker has thestructure:

wherein α represents the point of connection to the 3′-oxygen; wherein Rrepresents one or more atoms through which a covalent connection isestablished to the detectable label; and wherein Label is the detectablelabel.

In a further embodiment, the t-Butyldithiomethyl linker has thestructure:

wherein α represents the point of connection to the 3′-oxygen; wherein nis 1, 2, 3, 4, or 5; and wherein R′ represents one or more atoms throughwhich a covalent connection is established to the detectable label.

In a further embodiment of each of the foregoing methods, nucleotideanalogue is a nucleotide triphosphate, a nucleotide tetraphosphate, anucleotide pentaphosphate, or a nucleotide hexaphosphate.

In a further embodiment of each of the foregoing methods, the nucleotideanalogue(s) comprise a deoxyribose. In a further method the polymeraseis a DNA polymerase and the nucleic acid is DNA. In a further method ofeach of the foregoing methods, the nucleotide analogue(s) comprise aribose. In a further embodiment of each of the foregoing methods, thepolymerase is a reverse transcriptase and the nucleic acid is RNA. In afurther embodiment of each of the foregoing methods, the polymerase is aDNA-based RNA polymerase and the nucleic acid is DNA. In a furtherembodiment of each of the foregoing methods, the polymerase is anRNA-based RNA polymerase and the nucleic acid is RNA.

In a further embodiment, the t-Butyldithiomethyl linker has thestructure:

wherein α represents the point of connection to the 3′-oxygen; wherein Rrepresents one or more atoms through which a covalent connection isestablished to the detectable label; and wherein Label is the detectablelabel.

In a further embodiment, the detectable label is selected from the groupconsisting of a dye, a fluorophore, a combinatorial fluorescence energytransfer tag, a chemiluminescent compound, a chromophore, a mass tag, anelectrophore, a mononucleotide, an oligonucleotide, or a combinationthereof. In a further embodiment, the detectable label is a fluorophore.In a further embodiment, the fluorophore is selected from the groupconsisting of BodipyFL, R6G, ROX, Cy5, and Alexa488.

In a further embodiment, each nucleotide analog is selected from thegroup consisting of 3′-O-Alexa488-t-Butyldithiomethyl-dCTP,3′-O-Cy5-t-Butyldithiomethyl-dGTP, 3′-O-Rox-t-Butyldithiomethyl-dATP,3′-O-RG6-t-Butyldithiomethyl-dTTP,3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP,3-O-RG6-PEG₄-t-Butyldithiomethyl-dTTP,3′-O-Rox-PEG₄-t-Butyldithiomethyl-dATP,3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP.

In a further embodiment, the structure of each labeled nucleotide analogis selected from:

In a further embodiment, herein disclosed is a method of sequencing anucleic acid of interest which comprises repeatedly determining theidentity of each nucleotide present in the nucleic acid of interestaccording to any of the foregoing methods the method.

In a further embodiment of each of the foregoing sequencing methods,sequencing occurs simultaneously with a plurality of different nucleicacids of interest which comprises simultaneously sequencing each suchnucleic acid.

In a further embodiment of each of the foregoing methods, nucleotideanalogue is a nucleotide triphosphate, a nucleotide tetraphosphate, anucleotide pentaphosphate, or a nucleotide hexaphosphate.

In a further embodiment of each of the foregoing methods, the nucleotideanalogue(s) comprise a deoxyribose. In a further embodiment thepolymerase is a DNA polymerase and the nucleic acid is DNA. In a furtherembodiment of each of the foregoing methods, the nucleotide analogue(s)comprise a ribose. In a further embodiment of each of the foregoingmethods, the polymerase is a reverse transcriptase and the nucleic acidis RNA. In a further embodiment of each of the foregoing methods, thepolymerase is a DNA-based RNA polymerase and the nucleic acid is DNA. Ina further embodiment of each of the foregoing methods, the polymerase isan RNA-based RNA polymerase and the nucleic acid is RNA.

In a further embodiment of each of the foregoing methods, the nucleicacid of interest is immobilized on a solid support. In a furtherembodiment, the nucleic acid of interest is immobilized on the solidsupport via a 1,3-dipolar cycloaddition linkage, an amide bond or abiotin-streptavidin interaction. In a further embodiment, the solidsupport is in the form of a chip, a bead, a well, a capillary tube, or aslide. In a further embodiment, the solid support comprises gold,quartz, silica, or a plastic. In a further method, the solid support isporous.

In certain embodiments, the polymerase, single-stranded polynucleotide,DNA, or primer is bound to a solid substrate via 1,3-dipolarazide-alkyne cycloaddition chemistry. In an embodiment the polymerase,DNA, RNA, or primer, is bound to the solid substrate via a polyethyleneglycol molecule. In an embodiment the polymerase, DNA, RNA, or primer,is alkyne-labeled. In an embodiment the polymerase, DNA, RNA, or primer,is bound to the solid substrate via a polyethylene glycol molecule andthe solid substrate is azide-functionalized. In an embodiment thepolymerase, DNA, RNA, or primer, is immobilized on the solid substratevia an azido linkage, an alkynyl linkage, or biotin-streptavidininteraction. Immobilization of nucleic acids is described inImmobilization of DNA on Chips II, edited by Christine Wittmann (2005),Springer Verlag, Berlin, which is hereby incorporated by reference. Inan embodiment the DNA is single-stranded polynucleotide. In anembodiment the RNA is single-stranded RNA.

In other embodiments, the solid substrate is in the form of a chip, abead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, aporous media, a porous nanotube, or a column. This invention alsoprovides the any of the foregoing methods, wherein the solid substrateis a metal, gold, silver, quartz, silica, a plastic, polypropylene, aglass, or diamond. This invention also provides the instant method,wherein the solid substrate is a porous non-metal substance to which isattached or impregnated a metal or combination of metals. The solidsurface may be in different forms including the non-limiting examples ofa chip, a bead, a tube, a matrix, a nanotube. The solid surface may bemade from materials common for DNA microarrays, including thenon-limiting examples of glass or nylon. The solid surface, for examplebeads/micro-beads, may be in turn immobilized to another solid surfacesuch as a chip.

In one embodiment, the surface or substrate is a SERS-prepared surfaceor substrate designed specifically for detection of a label nucleotide.The surface may include one or more nanoplasmonic antenna, wherein thenanoplasmonic antenna may be a nanoplasmonic bowtie antenna. In oneembodiment, the nanoplasmonic bowtie antenna comprises crossed-bowtiestructure in which one pair of triangles couples to incident field,while another pair of triangles couples to Raman scattered field in anorthogonal polarization. It is also contemplated that the nanoplasmonicantenna may be an array of antennas. In addition, the nanoplasmonicantenna may include DNA functionalized sites, and may have a gap sizerange from 50 nm to 1 nm. In another embodiment, a nucleotide polymeraseis immobilized within the gap.

In another embodiment the nucleotide polymerase SERS-prepared anddesigned specifically for detection of a labeled nucleotide and/ornucleoside. The surface may include one or more nanoplasmonic antenna,wherein the nanoplasmonic antenna may be a nanoplasmonic bowtie antenna.In one embodiment, the nanoplasmonic bowtie antenna comprisescrossed-bowtie structure in which one pair of triangles couples toincident field, while another pair of triangles couples to Ramanscattered field in an orthogonal polarization. It is also contemplatedthat the nanoplasmonic antenna may be an array of antennas. In addition,the nanoplasmonic antenna may have a gap size range from 12 nm to 1 nm.In another embodiment, a nucleotide polymerase is immobilized within ona surface, substrate, or nanoplasmonic antenna on a surface.

In another embodiment, the surface comprises a DNA origami scaffold oran array of DNA origami scaffolds. It is also contemplated that the DNAorigami scaffold further comprising a primer molecules positionedbetween Au and Ag nanoparticles and nanorods located at specifiedbinding sites.

In a further embodiment, the surface comprises plasmonic crystals or anarray of plasmonic structures. For example, the plasmonic structures maybe periodic TiO—Au—TiO structures.

In various embodiments the polymerase, nucleic acid samples, DNA, RNA,or primer are separated in discrete compartments, wells or depressionson a surface.

In this invention methods are provided wherein about 1000 or fewercopies of the polymerase, nucleic acid sample, DNA, RNA, or primer arebound to the substrate. This invention also provides the instant methodswherein 2×10⁷, 1×10⁷, 1×10⁶ or 1×10⁴ or fewer copies of the polymerase,nucleic acid sample, DNA, RNA, or primer are bound to the substrate orsurface.

In further embodiments of the foregoing methods, the nucleotideincorporation events may be detected in real-time (i.e., as they occur).

Further embodiments of the foregoing methods may be single-moleculemethods. That is, the signal that is detected is generated by a singlemolecule (i.e., single nucleotide incorporation) and is not generatedfrom a plurality of clonal molecules. The methods may not require DNAamplification.

In other embodiments of the foregoing methods, a plurality of identicalsingle-stranded DNA or RNA molecules are sequenced simultaneously,thereby producing an aggregate signal.

In further embodiments of the foregoing methods, the signal generated bya nucleotide incorporation event is detected and/or generated throughthe use of a nanopore. Such nanopore devices and systems of the presentdisclosure may be combined with, or modified by other nanopore devicesand methods such as those described in U.S. Pat. Nos. 7,005,264 B2;7,846,738; 6,617,113; 6,746,594; 6,673,615; 6,627,067; 6,464,842;6,362,002; 6,267,872; 6,6015,714; 5,795,782; and U.S. Publication Nos.2015/0111759 and 2015/0368710, each of which is entirely incorporatedherein by reference.

In some embodiments, the immobilized polymerase, nucleic acid sample,DNA, RNA, or primer, is immobilized at a high density. This inventionalso provides the instant methods wherein over or up to 1×10⁷, 1×10⁸,1×10⁹ copies of the polymerase, nucleic acid sample, DNA, RNA, or primerare bound to the substrate or surface.

In other embodiments of the methods and/or compositions of thisinvention, the DNA is single-stranded. In other embodiments of themethods or of the compositions described herein, the single-strandedpolynucleotide is replaced with an RNA that is single-stranded.

Incorporation into an oligonucleotide or polynucleotide (such as aprimer or DNA extension strand) of a nucleotide and/or nucleoside analogmeans the formation of a phosphodiester bond between the 3′ carbon atomof the 3′ terminal nucleotide residue of the polynucleotide and the 5′carbon atom of the dNTP analog resulting in the loss of pyrophosphatefrom the dNTP analog.

A Raman spectroscopy system, as can be used in the methods describedherein, typically comprises an excitation source (such as a laser,including a laser diode in appropriate configuration, or two or morelasers), a sample illumination system and light collection optics, awavelength selector (such as a filter or spectrophotometer), and adetection apparatus (such as a CCD, a photodiode array, or aphotomultiplier). Interference (notch) filters with cut-off spectralrange of ±80-120 cm⁻¹ from the laser line can be used for stray lightelimination. Holographic gratings can be used. Double and triplespectrometers allow taking Raman spectra without use of notch filters.Photodiode Arrays (PDA) or a Charge-Coupled Devices (CCD) can be used todetect Raman scattered light.

In an embodiment, surface enhanced Raman spectroscopy (SERS) is usedwhich employs a surface treated with one or more of certain metals knownin the art to cause SERS effects. In an embodiment the surface is asurface to which the polymerase, polynucleotide, single-strandedpolynucleotide, single-stranded DNA polynucleotide, single-stranded RNA,primer, DNA extension strand, or oligonucleotide probe of the methodsdescribed herein is attached. Many suitable metals are known in the art.In an embodiment the surface is electrochemically etched silver ortreated with/comprises silver and/or gold colloids with average particlesize below 20 nm. The wavenumber of the Raman spectroscopy peak of anentity is identified by irradiating the entity with the excitationsource, such as a laser, and collecting the resulting Raman spectrumusing a detection apparatus. The wavenumber of the Raman spectroscopypeak is determined from the Raman spectrum. In an embodiment, thespectrum measured is from 2000 cm⁻¹ to 2300 cm⁻¹ and the wavenumber ofthe Raman spectroscopy peak is the peak wavenumber within that spectrum.In an embodiment the spectrum measured is a sub-range of 2000 cm⁻¹ to2300 cm⁻¹ and the Raman spectroscopy peak wavenumber is the peakwavenumber within that spectrum sub-range.

Where a range of values is provided, unless the context clearly dictatesotherwise, it is understood that each intervening integer of the value,and each tenth of each intervening integer of the value, unless thecontext clearly dictates otherwise, between the upper and lower limit ofthat range, and any other stated or intervening value in that statedrange, is encompassed within the invention. The upper and lower limitsof these smaller ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding (i) either or (ii)both of those included limits are also included in the invention.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

In choosing the compounds of the present invention, one of ordinaryskill in the art will recognize that the various substituents, i.e. R₁,R₂, etc. are to be chosen in conformity with well-known principles ofchemical structure connectivity.

In the compound structures depicted herein, hydrogen atoms, except onribose and deoxyribose sugars, are generally not shown. However, it isunderstood that sufficient hydrogen atoms exist on the representedcarbon atoms to satisfy the octet rule.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by a one of ordinary skillin the art to which this invention belongs.

As used herein, unless otherwise stated, the singular forms ‘a’, ‘an’,and ‘the’ include plural referents unless the context clearly dictatesotherwise. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as ‘solely’,‘only’ and the like in connection with the recitation of claim elements,or use of a ‘negative limitation’.

The methods described herein can be applied mutatis mutandis tosequencing RNA using the appropriate dNTPs and analogues thereof.

All combinations of the various elements described herein are within thescope of the invention. All sub-combinations of the various elementsdescribed herein are also within the scope of the invention. Eachembodiment disclosed herein is contemplated as being applicable to eachof the other disclosed embodiments. In addition, the elements recited inthe compound embodiments can be used in the composition and methodembodiments described herein and vice versa.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Embodiment P1

A nucleotide analog consisting of (i) a base, (ii) a sugar which may bea deoxyribose or a ribose, (iii) a t-butyldithiomethyl linker bound tothe 3′-oxygen of the deoxyribose or ribose, and (iv) a detectable labelbound to the t-butyldithiomethyl linker.

Embodiment P2

The nucleotide analog of embodiment P1, wherein the sugar is adeoxyribose.

Embodiment P3

The nucleotide analog of embodiment P1, wherein the sugar is a ribose.

Embodiment P4

The nucleotide analog of any one of embodiments P1-P3, wherein thenucleotide analog is a nucleotide monophosphate, a nucleotidediphosphate, a nucleotide triphosphate, a nucleotide tetraphosphate, anucleotide pentaphosphate, or a nucleotide hexaphosphate.

Embodiment P5

The nucleotide analog of any one of embodiments P1-P4, wherein the baseis adenine or an analog of adenine, guanine or an analog of guanine,cytosine or an analog of cytosine, thymine or an analog of thymine oruracil or an analog of uracil.

Embodiment P6

The nucleotide analog of any one of embodiments P1-P5, wherein thet-butyldithiomethyl linker has the structure:

wherein α represents the point of connection to the 3′-oxygen; wherein Rrepresents a structure consisting of one or more atoms one of which iscovalently bound to the detectable label; and wherein Label representsthe detectable label.

Embodiment P7

The nucleotide analog of embodiment P6, wherein the t-butyldithiomethyllinker has the structure:

wherein α represents the point of connection to the 3′-oxygen; wherein nis an integer which may be 1, 2, 3, 4, or 5; and wherein R′ represents astructure covalently attached to the detectable label.

Embodiment P8

The nucleotide analog of any one of embodiments P1-P7, wherein thedetectable label is a dye, a fluorophore, a fluorescence energy transfertag, a chemiluminescent compound, a chromophore, a mass tag, anelectrophore, a mononucleotide, an oligonucleotide, or a combinationthereof.

Embodiment P9

The nucleotide analog of embodiment P8, wherein the detectable label isa fluorophore.

Embodiment P10

The nucleotide analog of embodiment P9, wherein the fluorophore isBodipyFL, R6G, ROX, Cy5, or Alexa488.

Embodiment P11

The nucleotide analog of embodiment P1, wherein the nucleotide analog is3′-O-Alexa488-t-butyldithiomethyl-dCTP,3′-O-Cy5-t-butyldithiomethyl-dGTP, 3′-O-Rox-t-butyldithiomethyl-dATP,3′-O-RG6-t-butyldithiomethyl-dTTP,3′-O-Alexa488-PEG₄-t-butyldithiomethyl-dCTP,3′-O-RG6-PEG₄-t-butyldithiomethyl-dTTP,3′-O-Rox-PEG₄-t-butyldithiomethyl-dATP, or3′-O-Cy5-PEG₄-t-butyldithiomethyl-dGTP.

Embodiment P12

The nucleotide analog of embodiment P1, having the structure:

Embodiment P13

A composition comprising at least two different nucleotide analogs ofany one of embodiments 1-12, wherein each nucleotide analog consists ofa different base and a different detectable label from each othernucleotide analog present in the composition.

Embodiment P14

A method for determining the identity of a nucleotide at a predeterminedposition in a nucleic acid of interest, comprising:

-   -   a) providing        -   1) the nucleic acid of interest,        -   2) a nucleic acid polymerase.        -   3) a primer capable of hybridizing to said nucleic acid            immediately 3′ of such predetermined position,        -   4) four different nucleotide analogs of embodiment 1, each            of which consists of one of adenine or an analog of adenine,            guanine or an analog of guanine, cytosine or an analog of            cytosine, thiamine or an analog of thiamine, and a unique            detectable label;    -   b) incorporating one of said nucleotide analogs onto the end of        said primer to form an extension strand;    -   c) detecting the unique detectable label of the incorporated        nucleotide analog so as to thereby identify the incorporated        nucleotide analog on the end of said extension strand; and    -   d) based on the identity of the incorporated nucleotide,        determining the identity of the nucleotide at the predetermined        position.

Embodiment P15

The method of embodiment P14 further comprising, treating the extensionstrand of step (b) so as to cleave the t-butyldithiomethyl linker boundto the 3′-oxygen of the sugar and so as to produce a 3′-OH on the sugarand for producing an extension, remove the label from the extensionstrand to which another nucleotide analog may be added.

Embodiment P16

The method of any one of embodiments P14-P15, wherein treatmentcomprises contacting the extension strand withtris-(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine(THP).

Embodiment P17

The method of any one of embodiments P14-P16, wherein each nucleotideanalog is a nucleotide triphosphate, a nucleotide tetraphosphate, anucleotide pentaphosphate, or a nucleotide hexaphosphate.

Embodiment P18

The method of any one of embodiments P14-P17, wherein the nucleotideanalog comprises a deoxyribose.

Embodiment P19

The method of embodiment P18, wherein the polymerase is a DNA polymeraseand the nucleic acid is DNA.

Embodiment P20

The method of embodiment P18, wherein the polymerase is a reversetranscriptase and the nucleic acid is RNA.

Embodiment P21

The method of any one of embodiments P14-P20, wherein the nucleotideanalog comprises a ribose.

Embodiment P22

The method of embodiment P21, wherein the polymerase is a DNA-based RNApolymerase and the nucleic acid is DNA.

Embodiment P23

The method of embodiment P21, wherein the polymerase is an RNA-based RNApolymerase and the nucleic acid is RNA.

Embodiment P24

The method of any one of embodiments P14-P23, wherein thet-Butyldithiomethyl linker has the structure:

wherein α represents the point of connection to the 3′-oxygen; wherein Rrepresents one or more atoms through which a covalent connection isestablished to the detectable label; and wherein Label is the detectablelabel.

Embodiment P25

The method of any one of embodiments P14-P24, wherein thet-Butyldithiomethyl linker has the structure:

wherein α represents the point of connection to the 3′-oxygen; wherein nis 1, 2, 3, 4, or 5; and wherein R′ represents one or more atoms throughwhich a covalent connection is established to the detectable label.

Embodiment P26

The method of any one of embodiments P14-P25, wherein the detectablelabel is selected from the group consisting of a dye, a fluorophore, acombinatorial fluorescence energy transfer tag, a chemiluminescentcompound, a chromophore, a mass tag, an electrophore, a mononucleotide,an oligonucleotide, or a combination thereof.

Embodiment P27

The method of embodiment P26, wherein the detectable label is afluorophore.

Embodiment P28

The method of embodiment P27, wherein the fluorophore is selected fromthe group consisting of BodipyFL, R6G, ROX, Cy5, and Alexa488.

Embodiment P29

The method of any one of embodiments P14-P23, wherein each nucleotideanalog is selected from the group consisting of3′-O-Alexa488-t-Butyldithiomethyl-dCTP,3′-O-Cy5-t-Butyldithiomethyl-dGTP, 3′-O-Rox-t-Butyldithiomethyl-dATP,3′-O-RG6-t-Butyldithiomethyl-dTTP,3′-O-Alexa488-PEG4-t-Butyldithiomethyl-dCTP,3′-O-RG6-PEG4-t-Butyldithiomethyl-dTTP,3′-O-Rox-PEG4-t-Butyldithiomethyl-dATP, 3′-O-Cy5-PEG4-t-Butyldithiomethyl-dGTP.

Embodiment P30

The method of any one of embodiments P14-P23, wherein the structure ofeach labeled nucleotide analog is selected from:

Embodiment P31

The method of any one of embodiments P14-P30, wherein the nucleic acidof interest is immobilized on a solid support.

Embodiment P32

The method of embodiment P31, wherein the nucleic acid of interest isimmobilized on the solid support via an azido linkage, an alkynyllinkage, a 1,3-dipolar cycloaddition linkage, or a biotin-streptavidininteraction.

Embodiment P33

The method of any one of embodiments P31-P32, wherein the solid supportis in the form of a chip, a bead, a well, a capillary tube, or a slide.

Embodiment P34

The method of any of embodiments P31-P33, wherein the solid supportcomprises gold, quartz, silica, or a plastic.

Embodiment P35

The method of any of embodiments P31-P34, wherein the solid support isporous.

Embodiment P36

A method of sequencing a nucleic acid of interest which comprisesrepeatedly determining the identity of each nucleotide present in thenucleic acid of interest according to the method of any one ofembodiments P14-P35.

Embodiment P37

A method of simultaneously sequencing a plurality of different nucleicacids of interest which comprises simultaneously sequencing each suchnucleic acid according to the method of embodiment P36.

Embodiment P38

A process for producing a 3′-O-Bodipy-t-Butyldithiomethyl-dNTP,comprising:

-   -   a) reacting,        -   1) a 5′-O-tert-Butyldimethylsilyl-nucleoside,        -   2) acetic acid, and        -   3) acetic anhydride, under conditions permitting the            production of a            3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside:    -   b) contacting the        3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside        produced in step a) with trimethylamine, molecular sieve,        sulfuryl chloride, potassium p-toluenethiosulfonate, and        2,2,2,-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide, under        conditions permitting the production of a product having the        structure:

wherein B is a nucleobase

-   -   c) contacting the product produced in step b) with        tetrabutylammonium fluoride THF solution under conditions        permitting the production of a product having the structure:

-   -   -   wherein B is a nucleobase;

    -   d) contacting the product produced in step c) with        tetrabutylammonium pyrophosphate,        2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one, and iodine        solution under conditions permitting the production of a        3-O-NH₂-t-Butyldithiomethyl-dNTP;

    -   e) contacting the 3-O-NH₂-t-Butyldithiomethyl-dNTP of step d)        with Bodipy FL-NHS ester under conditions permitting the        production of the 3′-O-Bodipy-t-Butyldithiomethyl-dNTP.

Embodiment P39

The process of embodiment P38, wherein the3′-O-Bodipy-t-Butyldithiomethyl-dNTP is a3′-O-Bodipy-t-Butyldithiomethyl-dATP or an analog thereof,3′-O-Bodipy-t-Butyldithiomethyl-dTTP or an analog thereof,3′-O-Bodipy-t-Butyldithiomethyl-dGTP or an analog thereof, or3′-O-Bodipy-t-Butyldithiomethyl-dCTP.

Embodiment P40

The process of embodiment P39, wherein the3′-O-Bodipy-t-Butyldithiomethyl-dNTP is3′-O-Bodipy-t-Butyldithiomethyl-dTTP.

Embodiment P41

A process for producing a 3′-O-Bodipy-PEG₄-t-Butyldithiomethyl-dNTP,comprising:

-   -   a) reacting,        -   1) a 5′-O-tert-Butyldimethylsilyl-nucleoside,        -   2) acetic acid, and        -   3) acetic anhydride, under conditions permitting the            production of a            3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside:    -   b) contacting the        3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside        produced in part a) with trimethylamine, molecular sieve,        sulfuryl chloride, potassium p-toluenethiosulfonate, and        2,2,2,-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide, under        conditions permitting the production of a product having the        structure:

-   -   -   wherein B is a nucleobase:

    -   c) contacting the product produced in step b) with        tetrabutylammonium fluoride THF solution under conditions        permitting the production of a product having the structure:

-   -   -   wherein B is a nucleobase:

    -   d) contacting the product produced in step c) with        tetrabutylammonium pyrophosphate,        2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one, and iodine        solution under conditions permitting the production of a        3-O-NH₂-t-Butyldithiomethyl-dNTP;

    -   e) contacting the 3-O-NH₂-t-Butyldithiomethyl-dNTP of step d)        with Bodipy-PEG₄-Acid, N,N-disuccinimidyl carbonate, and        4-dimethylaminopyridine under conditions permitting the        production of the 3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dNTP.

Embodiment P42

The process of embodiment 41, wherein the3′-O-PEG4-Bodipy-t-Butyldithiomethyl-dNTP is a3′-O-PEG4-Bodipy-t-Butyldithiomethyl-dATP or an analog thereof,3′-O-PEG4-Bodipy-t-Butyldithiomethyl-dTTP or an analog thereof,3′-O-PEG4-Bodipy-t-Butyldithiomethyl-dGTP or an analog thereof, or3′-O-PEG4-Bodipy-t-Butyldithiomethyl-dCTP.

Embodiment P43

The process of embodiment 42, wherein the3′-O-PEG4-Bodipy-t-Butyldithiomethyl-dNTP is3′-O-PEG4-Bodipy-t-Butyldithiomethyl-dTTP.

Embodiment P44

A process for producing a 3′-O-Rox-t-Butyldithiomethyl-dATP, comprising:

-   -   a) reacting,        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine,            and        -   2) acetic acid and acetic anhydride            -   under conditions permitting the formation of a                N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of 3′-O-NH₂-t-Butyldithiomethyl-dATP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dATP produced in        step d) with ROX-NHS ester under conditions permitting the        production of the 3′-O-Rox-t-Butyldithiomethyl-dATP.

Embodiment P45

A process for producing a 3′-O-Rox-PEG₄-t-Butyldithiomethyl-dATP,comprising:

-   -   a) reacting.        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine,            and        -   2) acetic acid and acetic anhydride        -   under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine:    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of 3′-O-NH₂-t-Butyldithiomethyl-dATP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dATP produced in        step d) with ROX-PEG₄-Acid, N,N-disuccinimidyl carbonate, and        4-dimethylaminopyridine under conditions permitting the        production of the 3′-O-Rox-PEG₄t-Butyldithiomethyl-dATP.

Embodiment P46

A process for producing a 3′-O-Alexa488-t-Butyldithiomethyl-dCTP,comprising:

-   -   a) reacting        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine,            and        -   2) acetic acid and acetic anhydride        -   under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of a 3′-O-NH₂-t-Butyldithiomethyl-dCTP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dCTP produced in        step d) with Alexa488-NHS ester under conditions permitting the        production of the 3′-O-Alexa488-t-Butyldithiomethyl-dCTP.

Embodiment P47

A process for producing a 3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP,comprising:

-   -   a) reacting        -   1) a            1-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine, and        -   2) acetic acid and acetic anhydride        -   under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2-deoxycytidine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, I-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of a 3′-O-NH₂-t-Butyldithiomethyl-dCTP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dCTP produced in        step d) with Alexa488-PEG₄-NHS ester, N,N-disuccinimidyl        carbonate, and 4-dimethylaminopyridine under conditions        permitting the production of the        3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP.

Embodiment P48

A process for producing a 3′-O-Cy5-t-Butyldithiomethyl-dGTP, comprising:

-   -   a) reacting        -   1) a 2′-deoxyguanosine, and        -   2) tert-butyldimethylsilyl chloride, imidazole, and            N,N-dimethylformamide dimethyl acetal,        -   under conditions permitting the formation of a            N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine;    -   b) contacting the        N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine produced        in step a) with acetic acid and acetic anhydride under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with trimethylamine,        molecular sieves, sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   d) contacting the product of step c) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   e) contacting product of step d) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of 3′-O-NH₂-t-Butyldithiomethyl-dGTP;    -   f) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dGTP produced in        step e) with Cy5-NHS under conditions permitting the production        of the 3′-O-Cy5-t-Butyldithiomethyl-dGTP.

Embodiment P49

A process for producing a 3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP,comprising:

-   -   a) reacting        -   1) a 2′-deoxyguanosine, and        -   2) tert-butyldimethylsilyl chloride, imidazole, and            N,N-dimethylformamide dimethyl acetal,        -   under conditions permitting the formation of a            MN-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine;    -   b) contacting the        N-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine produced in        step a) with acetic acid and acetic anhydride under conditions        permitting the production of a product having the structure:

-   -   c) contacting the product of step b) with trimethylamine,        molecular sieves, sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   d) contacting the product of step c) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   e) contacting product of step d) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of 3′-O-NH₂-t-Butyldithiomethyl-dGTP;    -   f) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dGTP produced in        step e) with Cy5-PEG₄-NHS under conditions permitting the        production of the 3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP.

Embodiment R1

A nucleotide analogue comprised of (i) a base (ii) a sugar, and (iii) acleavable t-butyldithiomethyl moiety covalently attached to a 3′-oxygenof the sugar.

Embodiment R2

The nucleotide analog of embodiment R1, wherein the sugar is adeoxyribose.

Embodiment R3

The nucleotide analog of embodiment R1, wherein the sugar is a ribose.

Embodiment R4

The nucleotide analog of any one of embodiments R1-R3, wherein thenucleotide analog is a nucleotide monophosphate, a nucleotidediphosphate, a nucleotide triphosphate, a nucleotide tetraphosphate, anucleotide pentaphosphate, or a nucleotide hexaphosphate.

Embodiment R5

The nucleotide analogue of any one of embodiments R1-R4, wherein thebase is adenine or an analogue of adenine, guanine or an analogue ofguanine, cytosine or an analogue of cytosine, thymine or an analogue ofthymine, or uracil or an analogue of uracil.

Embodiment R6

The nucleotide analogue of any one of embodiments R1-R5, wherein thecleavable t-butyldithiomethyl moiety has the structure:

-   -   wherein α represents the point of connection to the 3′-oxygen.

Embodiment R7

The nucleotide analogue of embodiment R7, wherein the cleavablet-butyldithiomethyl moiety has the structure:

-   -   wherein α represents the point of connection to the 3′-oxygen;        and    -   wherein n is an integer which may be 1, 2, 3, 4, or 5.

Embodiment R8

The nucleotide analogue of embodiment 6, wherein the nucleotide analoguehas the structure:

Embodiment R9

The nucleotide analogue of any one of embodiments R1-R8, furthercomprising a detectable label.

Embodiment R10

The nucleotide analogue of embodiment R9, wherein the cleavablet-butyldithiomethyl moiety has the structure:

-   -   wherein α represents the point of connection to the 3′-oxygen;    -   wherein R represents a structure consisting of one or more atoms        one of which is covalently bound to the detectable label; and        wherein Label represents the detectable label.

Embodiment R11

The nucleotide analog of embodiment R10, wherein the cleavablet-butyldithiomethyl moiety has the structure:

-   -   wherein α represents the point of connection to the 3′-oxygen;    -   wherein n is an integer which may be 1, 2, 3, 4, or 5; and    -   wherein R′ represents one or more atoms through which a covalent        connection is established to the detectable label.

Embodiment R12

The nucleotide analogue of any one of embodiments R9-R11, wherein thenucleotide analogue has the structure:

-   -   wherein Cleavable Moiety is the cleavable t-butyldithiomethyl        moiety,    -   wherein Label represents the detectable label, and    -   wherein R′ represents one or more atoms through which a covalent        connection is established to the detectable label.

Embodiment R13

The nucleotide analog of any one of embodiments R9-R12, wherein thedetectable label is a dye, a fluorophore, a fluorescence energy transfertag, a chemiluminescent compound, a chromophore, a mass tag, anelectrophore, a mononucleotide, an oligonucleotide, or a combinationthereof.

Embodiment R14

The nucleotide analog of embodiment R13, wherein the detectable label isa fluorophore.

Embodiment R15

The nucleotide analog of embodiment R14, wherein the fluorophore isBodipyFL, R6G, ROX, Cy5, or Alexa488.

Embodiment R16

The nucleotide analog of embodiment R15, wherein the nucleotide analogis 3′-O-Alexa488-t-butyldithiomethyl-dCTP,3′-O-Cy5-t-butyldithiomethyl-dGTP, 3′-O-Rox-t-butyldithiomethyl-dATP,3′-O-RG6-t-butyldithiomethyl-dTTP,3′-O-Alexa488-PEG₄-t-butyldithiomethyl-dCTP,3′-O-RG6-PEG₄-t-butyldithiomethyl-dTTP,3′-O-Rox-PEG₄-t-butyldithiomethyl-dATP, or3′-O-Cy5-PEG₄-t-butyldithiomethyl-dGTP.

Embodiment R17

The nucleotide analog of embodiment R15, having the structure:

Embodiment R18

A composition comprising at least two different nucleotide analogues ofany one of embodiments R11-R17, wherein each nucleotide analogueconsists of a different base, and wherein each nucleotide analogueconsists of a different detectable label from each other nucleotideanalogue in the composition.

Embodiment R19

The nucleotide analogues of any one of embodiments R1-R9, furthercomprising an anchor moiety, wherein the anchor moiety is apredetermined small chemical moiety correlated to the identity of thebase and that orthogonally and rapidly reacts with a complementarybinding molecule thereby forming a conjugate of the anchor moiety andbinding molecule.

Embodiment R20

The nucleotide of embodiment R19, having the structure:

-   -   wherein base is one of adenine, guanine, thymine, cytosine,        uracil; or analogues thereof,    -   wherein Cleavable Moiety is the cleavable t-butyldithiomethyl        moiety,    -   wherein Anchor is the anchor moiety, and    -   wherein ω represents a structure consisting of one or more atoms        of which is covalently bound to both the t-butyldithiomethyl        cleavable moiety and the anchor moiety.

Embodiment R21

The nucleotide analogue of embodiment R20 or embodiment R21, wherein theanchor moiety has the structure:

-   -   wherein ω represents one or more atoms through which a covalent        connection is established to the cleavable t-butyldithiomethyl        moiety.

Embodiment R22

The nucleotide analogue of any one of embodiments R19-R21, wherein theanchor orthogonally and rapidly reacts with a complementary bindingmolecule thereby binding the anchor and binding molecule so as to form aconjugate of the anchor moiety and the binding molecule, wherein thebinding molecule has the structure:

-   -   wherein Label is a detectable label, and    -   wherein binder is a small chemical group correlated to the        identity of the detectable label and that orthogonally and        rapidly reacts with an anchor moiety thereby forming a conjugate        of the anchor moiety and binding molecule.

Embodiment R23

The nucleotide analogue of embodiment R22, wherein the detectable labelof the complementary binding molecule is selected from the groupconsisting of one or more dyes, fluorophores, combinatorial fluorescenceenergy transfer tags, chemiluminescent compounds, chromophores, masstags, electrophores, mononucleotides, oligonucleotides, or combinationsthereof.

Embodiment R24

The nucleotide analogue of embodiment R23, wherein the detectable labelof the complementary binding molecule comprises one or more fluorescenceenergy transfer tags.

Embodiment R25

The nucleotide analogue of embodiment R24, wherein the complementarybinding molecule further comprises one or more FRET cassettes.

Embodiment R26

The nucleotide analogue of embodiment R25, wherein the FRET cassettescomprise one or more dSpacer monomers.

Embodiment R27

The nucleotide analogue of embodiment R26, wherein the complementarybinding molecule has the structure:

-   -   wherein T1 is a point of attachment for one or more fluorescent        energy donor or acceptor, and T2 is a point of attachment for        one or more of the complementary energy donor or acceptor to        that in Ti, wherein n is an integer between 1 and 20, and R        represents the point of attachment to the binder of the binding        molecule.

Embodiment R28

The nucleotide analogue of embodiment R24, wherein the detectable labelof the complementary binding molecule is one or more fluorophore.

Embodiment R29

The nucleotide analogue of embodiment R28, wherein the fluorophore ofthe detectable label of the complementary binding molecule is selectedfrom the group consisting of BodipyFL, R6G, ROX, Cy5, and Alexa488.

Embodiment R30

The nucleotide analogue of any one of embodiments R22-R29, wherein thebinder of the complementary binding molecule comprises:

-   -   a) a compound comprising streptavidin having the structure:

-   -   b) a compound comprising the structure:

-   -   wherein α represents one or more atoms through which a covalent        connection is established to the detectable label.

Embodiment R31

The nucleotide analogue of embodiment 30, wherein the anchor moiety hasthe structure:

-   -   wherein ω represents one or more atoms through which a covalent        connection is established to the cleavable t-butyldithiomethyl        moiety, and    -   the anchor moiety orthogonally and rapidly reacts with a binder        of the complimentary binding molecule, wherein said binder        comprises streptavidin, and has the structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to the detectable label, and        -   thereby forms a conjugate having the structure:

-   -   -   -   wherein α is one or more atoms through which a covalent                connection is established to the detectable label, and                wherein ω is one or more atoms through which a covalent                connection is established to the cleavable                t-butyldithiomethyl moiety.

Embodiment R32

The nucleotide analogue of embodiment R31 having the structure:

-   -   wherein base is one of adenine, guanine, thymine, cytosine,        uracil, or analogues thereof.

Embodiment R33

The nucleotide analogue of embodiment R31 or R32, wherein the binder ofthe complementary binding molecule comprises streptavidin, and whereinthe complementary binding molecule has the structure:

Embodiment R34

The nucleotide analogue embodiment R30, wherein the anchor moiety hasthe structure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety,and the anchor moiety orthogonally and rapidly reacts with the binder ofthe complimentary binding molecule, wherein said binder has thestructure:

wherein α is one or more atoms through which a covalent connection isestablished to the detectable label, and thereby forms a conjugatehaving the structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to the detectable label, and wherein ω        is one or more atoms through which a covalent connection is        established to the cleavable t-butyldithiomethyl moiety.

Embodiment R35

The nucleotide analogue of embodiment R34 having the structure:

-   -   wherein base is one of adenine, guanine, thymine, cytosine,        uracil, or P an analogue thereof.

Embodiment R36

The nucleotide analogue of any one of embodiments R34-R35, wherein thecomplementary binding molecule has the structure:

Embodiment R37

The nucleotide analogue of embodiment R30, wherein the anchor moiety hasthe structure:

-   -   wherein ω represents one or more atoms through which a covalent        connection is established to the cleavable t-butyldithiomethyl        moiety, and        -   the anchor moiety orthogonally and rapidly reacts with the            binder of the complimentary binding molecule, wherein said            binder has the structure:

-   -   -   wherein α is one or more atoms through which a covalent            connection is established to the detectable label, and

    -   thereby forms a conjugate having the structure:

-   -   -   wherein α is one or more atoms through which a covalent            connection is established to the detectable label, and            wherein ω is one or more atoms through which a covalent            connection is established to the cleavable            t-butyldithiomethyl moiety.

Embodiment R38

The nucleotide analogue of embodiment R37 having the structure:

-   -   wherein base is one of adenine, guanine, thymine, cytosine,        uracil, or derivatives thereof.

Embodiment R39

The nucleotide analogue of any one of embodiments R37-R38, wherein thecomplementary binding molecule has the structure:

Embodiment R40

The nucleotide analogue of embodiment R30, wherein the anchor has thestructure:

wherein ω represents one or more atoms through which a covalentconnection is established to the cleavable t-butyldithiomethyl moiety,and the anchor orthogonally and rapidly reacts with a binder of acomplimentary binding molecule, wherein said binder has the structure:

wherein α is one or more atoms through which a covalent connection isestablished to a detectable label, and thereby forms a conjugate havingthe structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to detectable label, and wherein ω is        one or more atoms through which a covalent connection is        established to the cleavable t-butyldithiomethyl moiety.

Embodiment R41

The nucleotide analogue of embodiment R40, wherein the nucleotideanalogue has the structure

wherein base is one of adenine, guanine, thymine, cytosine, uracil, oran analogue thereof.

Embodiment R42

The nucleotide analogue of any one of embodiments R40-R41, wherein thecomplementary binding molecule has the structure:

Embodiment R43

The nucleotide analogue of any one of embodiments R1-R42, wherein thecleavable t-butyldithiomethyl moiety may be cleaved by a water solublephosphine, thereby resulting in a 3′-OH.

Embodiment R44

The nucleotide analogue of embodiment R43, wherein the water solublephosphine is tris-(2-carboxyethyl)phosphine (TCEP) ortris(hydroxypropyl)phosphine (THP).

Embodiment R45

A composition comprising at least two nucleotide analogues of any one ofembodiments R1-R44, wherein each nucleotide analogue has a differentbase.

Embodiment R46

A composition comprising at least two nucleotide analogues of any one ofembodiments 19-44, wherein each nucleotide analogue has a differentbase, and wherein each nucleotide analogue has a different anchormoiety.

Embodiment R47

A process for producing a 3′-O-Bodipy-t-Butyldithiomethyl-dNTP,comprising:

-   -   a) reacting,        -   1) a 5′-O-tert-Butyldimethylsilyl-nucleoside,        -   2) acetic acid, and        -   3) acetic anhydride, under conditions permitting the            production of a            3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside;    -   b) contacting the        3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside        produced in step a) with trimethylamine, molecular sieve,        sulfuryl chloride, potassium p-toluenethiosulfonate, and        2,2,2,-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide, under        conditions permitting the production of a product having the        structure:

-   -   -   wherein B is a nucleobase;

    -   c) contacting the product produced in step b) with        tetrabutylammonium fluoride THF solution under conditions        permitting the production of a product having the structure:

-   -   -   wherein B is a nucleobase:

    -   d) contacting the product produced in step c) with        tetrabutylammonium pyrophosphate,        2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one, and iodine        solution under conditions permitting the production of a        3-O-NH₂-t-Butyldithiomethyl-dNTP;

    -   e) contacting the 3-O-NH₂-t-Butyldithiomethyl-dNTP of step d)        with Bodipy FL-NHS ester under conditions permitting the        production of the 3′-O-Bodipy-t-Butyldithiomethyl-dNTP

Embodiment R48

The process of embodiment R47, wherein the3′-O-Bodipy-t-Butyldithiomethyl-dNTP is a3′-O-Bodipy-t-Butyldithiomethyl-dATP or an analog thereof,3′-O-Bodipy-t-Butyldithiomethyl-dTTP or an analog thereof,3′-O-Bodipy-t-Butyldithiomethyl-dGTP or an analog thereof, or3′-O-Bodipy-t-Butyldithiomethyl-dCTP.

Embodiment R49

The process of embodiment R48, wherein the3′-O-Bodipy-t-Butyldithiomethyl-dNTP is3′-O-Bodipy-t-Butyldithiomethyl-dTTP.

Embodiment R50

A process for producing a 3′-O-Bodipy-PEG₄-t-Butyldithiomethyl-dNTP,comprising:

-   -   a) reacting,        -   1) a 5′-O-tert-Butyldimethylsilyl-nucleoside,        -   2) acetic acid, and        -   3) acetic anhydride,    -   under conditions permitting the production of a        3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside;    -   b) contacting the        3′-O-methylthiomethyl-5′-O-tert-butyldimerthylsilyl-nucleoside        produced in part a) with trimethylamine, molecular sieve,        sulfuryl chloride, potassium p-toluenethiosulfonate, and        2,2,2,-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide, under        conditions permitting the production of a product having the        structure:

-   -   -   wherein B is a nucleobase:

    -   c) contacting the product produced in step b) with        tetrabutylammonium fluoride THF solution under conditions        permitting the production of a product having the structure:

-   -   -   wherein B is a nucleobase:

    -   d) contacting the product produced in step c) with        tetrabutylammonium pyrophosphate,        2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one, and iodine        solution under conditions permitting the production of a        3-O-NH₂-t-Butyldithiomethyl-dNTP;

    -   e) contacting the 3-O-NH₂-t-Butyldithiomethyl-dNTP of step d)        with Bodipy-PEG₄-Acid, N,N-disuccinimidyl carbonate, and        4-dimethylaminopyridine under conditions permitting the        production of the 3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dNTP.

Embodiment R51

The process of embodiment 50, wherein the3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dNTP is a3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dATP or an analog thereof,3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dTTP or an analog thereof,3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dGTP or an analog thereof, or3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dCTP.

Embodiment R52

The process of embodiment 51, wherein the3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dNTP is3′-O-PEG₄-Bodipy-t-Butyldithiomethyl-dTTP.

Embodiment R53

A process for producing a 3′-O-Rox-t-Butyldithiomethyl-dATP, comprising:

-   -   a) reacting,        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine,            and        -   2) acetic acid and acetic anhydride under conditions            permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of 3′-O-NH₂-t-Butyldithiomethyl-dATP; e)        contacting the 3′-O-NH₂-t-Butyldithiomethyl-dATP produced in        step d) with ROX-NHS ester under conditions permitting the        production of the 3′-O-Rox-t-Butyldithiomethyl-dATP.

Embodiment R54

A process for producing a 3′-O-Rox-PEG₄-t-Butyldithiomethyl-dATP,comprising:

-   -   a) reacting,        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine,            and        -   2) acetic acid and acetic anhydride        -   under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of 3′-O-NH₂-t-Butyldithiomethyl-dATP; e)        contacting the 3′-O-NH₂-t-Butyldithiomethyl-dATP produced in        step d) with ROX-PEG₄-Acid, N,N-disuccinimidyl carbonate, and        4-dimethylaminopyridine under conditions permitting the        production of the 3′-O-Rox-PEG₄t-Butyldithiomethyl-dATP.

Embodiment R55

A process for producing a 3′-O-Alexa488-t-Butyldithiomethyl-dCTP,comprising:

-   -   a) reacting        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine,            and        -   2) acetic acid and acetic anhydride        -   under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of a 3′-O-NH₂-t-Butyldithiomethyl-dCTP; e)        contacting the 3′-O-NH₂-t-Butyldithiomethyl-dCTP produced in        step d) with Alexa488-NHS ester under conditions permitting the        production of the 3′-O-Alexa488-t-Butyldithiomethyl-dCTP.

Embodiment R56

A process for producing a 3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP,comprising:

-   -   a) reacting        -   1) a            N⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine,            and        -   2) acetic acid and acetic anhydride        -   under conditions permitting the formation of a            N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine;    -   b) contacting the        N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine        produced in step a) with trimethylamine, molecular sieves,        sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   d) contacting product of step c) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of a 3′-O-NH₂-t-Butyldithiomethyl-dCTP;    -   e) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dCTP produced in        step d) with Alexa488-PEG₄-NHS ester, N,N-disuccinimidyl        carbonate, and 4-dimethylaminopyridine under conditions        permitting the production of the        3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP.

Embodiment R57

A process for producing a 3′-O-Cy5-t-Butyldithiomethyl-dGTP, comprising:

-   -   a) reacting        -   1) a 2′-deoxyguanosine, and        -   2) tert-butyldimethylsilyl chloride, imidazole, and            N,N-dimethylformamide dimethyl acetal,        -   under conditions permitting the formation of a            N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine:    -   b) contacting the        N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine produced        in step a) with acetic acid and acetic anhydride under        conditions permitting the production of a product having the        structure:

-   -   c) contacting the product of step b) with trimethylamine,        molecular sieves, sufuryl chloride, p-toluenethiosulfonate, and        2,2,2,-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   d) contacting the product of step c) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   e) contacting product of step d) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of 3′-O-NH₂-t-Butyldithiomethyl-dGTP;    -   f) contacting the 3′-O-NH₂-t-Butyldithiomethyl-dGTP produced in        step e) with Cy5-NHS under conditions permitting the production        of the 3′-O-Cy5-t-Butyldithiomethyl-dGTP.

Embodiment R58

A process for producing a 3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP,comprising:

-   -   a) reacting        -   1) a 2′-deoxyguanosine, and        -   2) tert-butyldimethylsilyl chloride, imidazole, and            N,N-dimethylformamide dimethyl acetal,        -   under conditions permitting the formation of a            N-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine;    -   b) contacting the        M-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine produced in        step a) with acetic acid and acetic anhydride under conditions        permitting the production of a product having the structure:

-   -   c) contacting the product of step b) with trimethylamine,        molecular sieves, sufuryl chloride, p-toluenethiosulfonate, and        2,2,2-trifluor-N-(2-mercapto-2-methylpropyl)acetamide under        conditions permitting the production of a product having the        structure:

-   -   d) contacting the product of step c) with tetrabutylammonium        fluoride THF solution under conditions permitting the production        of a product having the structure:

-   -   e) contacting product of step d) with tetrabutylammonium        pyrophosphate, 1-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,        tributylamine, and iodine solution under conditions permitting        the production of 3′-O-NH₂-t-Butyldithiomethyl-dGTP; f)        contacting the 3′-O-NH₂-t-Butyldithiomethyl-dGTP produced in        step e) with Cy5-PEG₄-NHS under conditions permitting the        production of the 3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP.

Embodiment R59

A method for determining the nucleotide sequence of a single-strandedDNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first        type of nucleotide analogue under conditions permitting the        nucleotide polymerase to catalyze incorporation of the        nucleotide analogue into the primer if the nucleotide analogue        is complementary to a nucleotide residue of the single-stranded        DNA that is immediately 5′ to a nucleotide residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, so as to form a DNA extension product,        wherein the nucleotide analogue has the structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein ω represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,        -   wherein the identity of the anchor moiety is predetermined            and is correlated to the identity of the base,

    -   b) contacting the single-stranded DNA of step a) with a binding        molecule complementary to the anchor moiety of the nucleotide        analogue of step a), wherein the binding molecule has the        structure:

-   -   -   wherein binder is a chemical group that orthogonally and            rapidly reacts with the anchor moiety, thereby forming a            conjugate of the binding molecule and the anchor moiety, and            Label is a detectable label.

    -   c) removing any nucleotide analogue not incorporated into the        primer in step a);

    -   d) detecting the presence of any detectable label so as to        thereby determine whether the nucleotide analogue of step a) was        incorporated so as to thereby determine the identity of the        complementary nucleotide residue in the single-stranded DNA, and        -   wherein if the base of the nucleotide analogue a) is not            complementary to the nucleotide residue of the            single-stranded DNA which is immediately 5′ to the            nucleotide residue of the single-stranded DNA hybridized to            the 3′ terminal nucleotide residue of the primer, then            iteratively repeating steps a) through c) with a second,            third, and then fourth type of nucleotide analogue, wherein            each different type of nucleotide analogue has a different            base from each other type of nucleotide analogue, until the            nucleotide analogue has a base that is complementary,

    -   e) cleaving the cleavable t-butyldithiomethyl moiety, so as to        thereby create a 3′-OH; and

    -   f) iteratively performing steps a) through e) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Embodiment R60

The method of embodiment R59, wherein steps b) and c) can be performedsimultaneously, or in the order step b) then step c) or in the orderstep c) then step b).

Embodiment R61

The method of embodiment R59 or embodiment R60, where the first, second,third, and fourth type of nucleotide analogue have different anchormoieties, and wherein each different anchor moiety is complementary to adifferent binding molecule.

Embodiment R62

The method of any one of embodiment R61, wherein the different bindingmolecules each have a different detectable label.

Embodiment R63

A method for determining the nucleotide sequence of a single-strandedDNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product, wherein each type of nucleotide analogue has the        structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein ω represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,        -   wherein the base of each type of nucleotide analogue is            independently different from the base of the remaining three            types of nucleotide analogue, wherein the anchor moiety of            each type of nucleotide analogue is independently different            from the anchor moiety of the remaining three types of            nucleotide analogue, wherein the anchor moiety of each type            of nucleotide analogue orthogonally and rapidly reacts with            a different binding molecule from each of the remaining            three types of nucleotide analogue;

    -   b) contacting the single-stranded DNA of step a) with a first,        second, third, and fourth type of binding molecule, under        conditions permitting the anchor of the nucleotide analogue        incorporated in step a) to orthogonally and rapidly react with a        complementary binding molecule thereby forming a conjugate of        the binding molecule and the anchor moiety,        -   wherein the first, second, third, and fourth type of binding            molecule each have the structure:

-   -   -   wherein binder is a small chemical group that orthogonally            and rapidly reacts with an anchor moiety, and wherein Label            is a predetermined detectable label correlated to the            identity of the type of binding molecule, wherein the binder            of each type of binding molecule is different from the            binder of the remaining three types of binding molecule,            wherein the first type of binding molecule and the first            type of nucleotide analogue, the second type of binding            molecule and second type of nucleotide analogue, the third            type of binding molecule and third type of nucleotide            analogue, and the fourth type of binding molecule and the            fourth type of nucleotide analogue are respectively            complementary and thereby orthogonally and rapidly react            thereby forming a conjugate of an individual type of binding            molecule with an individual type of nucleotide analogue;

    -   c) determining the identity of the detectable label of the        nucleotide analogue incorporated in step a) so as to thereby        determine the identity of the incorporated nucleotide analogue        and the identity of the complementary nucleotide residue in the        single-stranded DNA;

    -   d) cleaving the cleavable t-butyldithiomethyl moiety, so as to        thereby create a 3′-OH; and

    -   e) iteratively performing steps a) through d) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Embodiment R64

A method for determining the nucleotide sequence of a single-strandedDNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first and second types of nucleotide analogue            have the structure:

-   -   -   -   wherein base is one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein Linker                is a cleavable t-butyldithiomethyl moiety that when                bound to the 3′-O prevents a nucleotide polymerase from                catalyzing a polymerase reaction with the 3′-O of the                nucleotide analogue, wherein Label is a predetermined                detectable label, and wherein R represents a structure                consisting of one or more atoms of which is covalently                bound to both the cleavable t-butyldithiomethyl moiety                and the detectable label,            -   wherein the label of the first type of nucleotide                analogue is different form the label of the second type                of nucleotide analogue, wherein the base of each of the                first and second type of nucleotide analogue is                independently different from the base of each of the                three remaining types of nucleotide analogue,

        -   wherein the third and fourth type of nucleotide analogue has            the structure:

-   -   -   -   wherein base is any one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein                Cleavable Moiety is a cleavable t-butyldithiomethyl                moiety that when bound to the 3′-O prevents a nucleotide                polymerase from catalyzing a polymerase reaction with                the 3′-O of the nucleotide analogue, wherein Anchor is                an anchor moiety that is a small chemical moiety that                orthogonally and rapidly reacts with a complementary                binding molecule thereby forming a conjugate of the                anchor moiety and binding molecule, wherein ω represents                a structure consisting of one or more atoms of which is                covalently bound to both the cleavable                t-butyldithiomethyl moiety and the anchor moiety,            -   wherein the base of the third and fourth type of                nucleotide analogue is independently different from the                base of each of the three remaining types of nucleotide                analogue, wherein the anchor moiety of the third type of                nucleotide analogue is different from the anchor moiety                of the fourth type of nucleotide analogue;

    -   b) removing any nucleotide analogues not incorporated in step        a);

    -   c) detecting the presence of either the detectable label of the        first or second type of nucleotide analogue incorporated in        step a) so as to thereby determine the identity of the        incorporated nucleotide analogue and the identity of the        complementary nucleotide residue in the single-stranded DNA,        -   wherein if the base of the first and second type of            nucleotide is not complementary, contacting the            single-stranded DNA with a first and second type of binding            molecule, wherein the first and second type of binding            molecule have the structure:

-   -   -   -   wherein binder is a small chemical group that                orthogonally and rapidly reacts with an anchor, and                wherein Label is a predetermined detectable label                correlated to the identity of the binding molecule,                wherein the detectable label of the first type of                binding molecule is the same as the detectable label of                the first type of nucleotide analogue, wherein the                detectable label of the second type of binding molecule                is the same as the detectable label of the second type                of nucleotide analogue, wherein the binder of the first                type of binding molecule orthogonally and rapidly reacts                with the anchor of the third type of nucleotide                analogue, and wherein the second type of binding                molecule orthogonally and rapidly reacts with the anchor                of the fourth type of nucleotide analogue,

        -   removing any unbound binding molecule, and detecting the            presence of either the first or second binding molecule so            as to thereby determine the identity of the nucleotide            analogue incorporated in step a) and the identity of the            complementary nucleotide residue in the single-stranded DNA;

    -   d) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   e) iteratively performing steps a) through d) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Embodiment R65

A method for determining the nucleotide sequence of a single-strandedDNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first, second, and third type of nucleotide            analogue have the structure:

-   -   -   -   wherein base is any one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein                Cleavable Moiety is a cleavable t-butyldithiomethyl                moiety that when bound to the 3′-O prevents a nucleotide                polymerase from catalyzing a polymerase reaction with                the 3′-O of the nucleotide analogue, wherein Anchor is                an anchor moiety that is a small chemical moiety that                orthogonally and rapidly reacts with a complementary                binding molecule thereby forming a conjugate of the                anchor moiety and binding molecule, wherein ω represents                a structure consisting of one or more atoms of which is                covalently bound to both the cleavable                t-butyldithiomethyl moiety and the anchor moiety,            -   wherein the base of the first, second, and third type of                nucleotide analogue is independently different from the                base of each of the three remaining types of nucleotide                analogue, wherein the first, second, and third type of                nucleotide analogue each independently have a different                anchor from one another,

        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   -   wherein base is one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein                Cleavable Moiety is a cleavable t-butyldithiomethyl                moiety that when bound to the 3′-O prevents a nucleotide                polymerase from catalyzing a polymerase reaction with                the 3′-O of the nucleotide analogue, wherein the base of                the fourth type of nucleotide analogue is independently                different from the base of each of the three remaining                types of nucleotide analogue:

    -   b) contacting the single-stranded DNA of step a) with a first,        second, and third type of binding molecule, each type of binding        molecule having the structure:

-   -   -   wherein binder is a small chemical group correlated to the            identity of the type of binding molecule and that            orthogonally and rapidly reacts with an anchor so as to form            a conjugate, and wherein Label is a detectable label,        -   wherein the binder of each type of binding molecule is            different from the binder of the remaining two types of            binding molecule, wherein the first type of binding molecule            and the first type of nucleotide analogue, the second type            of binding molecule and second type of nucleotide analogue,            and third type of binding molecule and third type of            nucleotide analogue are respectively complementary and            thereby orthogonally and rapidly react thereby binding each            individual type of binding molecule with an individual type            of nucleotide analogue:

    -   c) removing any nucleotide analogues from step a) not        incorporated into the primer;

    -   d) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   e) if a detectable label is detected in step d), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the first type of        nucleotide analogue;

    -   f) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the first type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   g) if a detectable label is detected in step f), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the second type of        nucleotide analogue;

    -   h) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   i) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   j) iteratively performing steps a) through i) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Embodiment R66

A method for determining the nucleotide sequence of a single-strandedDNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,        -   wherein the first and second types of nucleotide analogue            have the structure:

-   -   -   -   wherein base is any one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein                Cleavable Moiety is a cleavable t-butyldithiomethyl                moiety that when bound to the 3′-O prevents a nucleotide                polymerase from catalyzing a polymerase reaction with                the 3′-O of the nucleotide analogue, wherein Anchor is                an anchor moiety that is a small chemical moiety that                orthogonally and rapidly reacts with a complementary                binding molecule thereby forming a conjugate of the                anchor moiety and binding molecule, wherein ω represents                a structure consisting of one or more atoms of which is                covalently bound to both the cleavable                t-butyldithiomethyl moiety and the anchor moiety,            -   wherein the base of the first and second type of                nucleotide analogue is independently different from the                base of each of the three remaining types of nucleotide                analogue, wherein the anchor of the first type of                nucleotide analogue is different from the anchor of the                second type of nucleotide analogue,

        -   wherein the third type of nucleotide analogue has the            structure:

-   -   -   -   wherein base is one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein Linker                is a cleavable t-butyldithiomethyl moiety that when                bound to the 3′-O prevents a nucleotide polymerase from                catalyzing a polymerase reaction with the 3′-O of the                nucleotide analogue, wherein Label is a predetermined                detectable label, and wherein R represents a structure                consisting of one or more atoms of which is covalently                bound to both the cleavable t-butyldithiomethyl moiety                and the detectable label,            -   wherein the base of the third type of nucleotide                analogue is independently different from the base of                each of the three remaining types of nucleotide                analogue,

        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   -   wherein base is one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein                Cleavable Moiety is a cleavable t-butyldithiomethyl                moiety that when bound to the 3′-O prevents a nucleotide                polymerase from catalyzing a polymerase reaction with                the 3′-O of the nucleotide analogue, wherein the base of                the fourth type of nucleotide analogue is independently                different from the base of each of the three remaining                types of nucleotide analogue:

    -   b) removing any nucleotide analogues from step a) not        incorporated into the primer;

    -   c) detecting whether there is a presence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the third type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   d) if an absence of detectable label bound to the incorporated        nucleotide of step a) is detected in step c), contacting the        single-stranded DNA with a first and second type of binding        molecule, wherein the first and second type of binding molecule        have the structure:

-   -   -   wherein binder is a small chemical group correlated to the            identity of the type of binding molecule and that            orthogonally and rapidly reacts with an anchor, and wherein            Label is a detectable label,        -   wherein the binder of each type of binding molecule is            different one another, wherein the first type of binding            molecule and the first type of nucleotide analogue, and the            second type of binding molecule and second type of            nucleotide analogue, respectively complementary and thereby            orthogonally and rapidly react thereby binding each            individual type of binding molecule with an individual type            of nucleotide analogue so as to form a conjugate;

    -   e) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   f) if a detectable label is detected in step e), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the first type of        nucleotide analogue;

    -   g) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   h) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   i) iteratively performing steps a) through h) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Embodiment R67

A method for determining the nucleotide sequence of a single-strandedDNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product,    -   wherein the first, second, and third type of nucleotide analogue        have the structure:

-   -   -   wherein base is any one of adenine, guanine, thymine,            cytosine, uracil, or an analogue thereof, wherein Cleavable            Moiety is a cleavable t-butyldithiomethyl moiety that when            bound to the 3′-O prevents a nucleotide polymerase from            catalyzing a polymerase reaction with the 3′-O of the            nucleotide analogue, wherein Anchor is an anchor moiety that            is a small chemical moiety that orthogonally and rapidly            reacts with a complementary binding molecule thereby forming            a conjugate of the anchor moiety and binding molecule,            wherein ω represents a structure consisting of one or more            atoms of which is covalently bound to both the cleavable            t-butyldithiomethyl moiety and the anchor moiety,        -   wherein the base of the first, second, and third type of            nucleotide analogue is independently different from the base            of each of the three remaining types of nucleotide analogue,            wherein the first, second, and third type of nucleotide            analogue each independently have a different anchor from one            another,        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Linker is a            cleavable t-butyldithiomethyl moiety that when bound to the            3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein Label is a predetermined detectable label,            and wherein R represents a structure consisting of one or            more atoms of which is covalently bound to both the            cleavable t-butyldithiomethyl moiety and the detectable            label,        -   wherein the base of the fourth type of nucleotide analogue            is independently different from the base of each of the            three remaining types of nucleotide analogue;

    -   b) removing all unincorporated nucleotide analogues from step        a);

    -   c) detecting whether there is a presence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   d) if an absence of detectable label bound to the incorporated        nucleotide of step a) is detected in step c), contacting the        single-stranded DNA with a first, second, and third type of        binding molecule, wherein the first, second, and third type of        binding molecule have the structure:

-   -   wherein binder is a small chemical group correlated to the        identity of the type of binding molecule and that orthogonally        and rapidly reacts with an anchor moiety so as to form a        conjugate, and wherein Label is a detectable label,    -   wherein the binder of each type of binding molecule is different        from one another, wherein the first type of binding molecule and        wherein the first type of binding molecule and the first type of        nucleotide analogue, the second type of binding molecule and        second type of nucleotide analogue, and third type of binding        molecule and third type of nucleotide analogue are respectively        complementary and thereby orthogonally and rapidly react thereby        binding each individual type of binding molecule with an        individual type of nucleotide analogue so as to form a        conjugate;    -   e) detecting whether there is a presence of detectable label        bound to the incorporated nucleotide of step a);    -   f) contacting the single-stranded DNA with a means of cleaving        the detectable label and/or the binding molecule from the first        type of nucleotide analogue;    -   g) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that the identity of the incorporated nucleotide is        of the first type of nucleotide analogue, and thereby the        identity of the complementary nucleotide residue in the        single-stranded DNA;    -   h) if a detectable label is detected in step f), contacting the        single-stranded DNA with a means of cleaving the detectable        label and/or the binding molecule from the second type of        nucleotide analogue;    -   i) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;    -   j) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and    -   k) iteratively performing steps a) through j) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Embodiment R68

A method for determining the nucleotide sequence of a single-strandedDNA, the method comprising:

-   -   a) contacting the single-stranded DNA having a primer hybridized        to a portion thereof, with a nucleotide polymerase and a first,        second, third, and fourth type of nucleotide analogue under        conditions permitting the nucleotide polymerase to catalyze        incorporation of a nucleotide analogue into the primer if the        nucleotide analogue is complementary to a nucleotide residue of        the single-stranded DNA that is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product.    -   wherein the first, second, and third types of nucleotide        analogue have the structure:

-   -   -   wherein base is one of adenine, guanine, thymine, cytosine,            uracil, or an analogue thereof, wherein Linker is a            cleavable t-butyldithiomethyl moiety that when bound to the            3′-O prevents a nucleotide polymerase from catalyzing a            polymerase reaction with the 3′-O of the nucleotide            analogue, wherein Label is a predetermined detectable label,            and wherein R represents a structure consisting of one or            more atoms of which is covalently bound to both the            cleavable t-butyldithiomethyl moiety and the detectable            label,        -   wherein the base of the first, second, and third type of            nucleotide analogue is independently different from the base            of each of the three remaining types of nucleotide analogue,        -   wherein the fourth type of nucleotide analogue has the            structure:

-   -   -   -   wherein base is one of adenine, guanine, thymine,                cytosine, uracil, or an analogue thereof, wherein                Cleavable Moiety is a cleavable t-butyldithiomethyl                moiety that when bound to the 3′-O prevents a nucleotide                polymerase from catalyzing a polymerase reaction with                the 3′-O of the nucleotide analogue, wherein the base of                the fourth type of nucleotide analogue is independently                different from the base of each of the three remaining                types of nucleotide analogue:

    -   b) removing all unincorporated nucleotide analogues from step        a);

    -   c) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the fourth type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   d) if a detectable label is detected in step c), contacting the        single-stranded DNA with a means of cleaving the detectable        label from the first type of nucleotide analogue;

    -   e) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining that identity of the incorporated nucleotide is of        the first type of nucleotide analogue, and thereby the identity        of the complementary nucleotide residue in the single-stranded        DNA;

    -   i) if a detectable label is detected in step e), contacting the        single-stranded DNA with a means of cleaving the detectable        label from the second type of nucleotide analogue;

    -   g) detecting whether there is an absence of detectable label        bound to the incorporated nucleotide of step a), thereby        determining the identity of the incorporated nucleotide        analogue, and thereby the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   h) cleaving the cleavable t-butyldithiomethyl moiety so as to        thereby create a 3′-OH; and

    -   i) iteratively performing steps a) through h) for each        nucleotide residue of the single-stranded DNA to be sequenced so        as to thereby determine the sequence of the single-stranded DNA.

Embodiment R69

The method of any one of embodiments R59-R67, wherein the anchor of eachtype of nucleotide analogue having an anchor that forms a conjugate witha complementary binding molecule, each individually has the structure:

-   -   wherein ω represents one or more atoms through which a covalent        connection is established to the cleavable t-butyldithiomethyl        moiety.

Embodiment R70

The method of embodiment R69, wherein the detectable label of thecomplementary binding molecule is selected from the group consisting ofone or more dyes, fluorophores, combinatorial fluorescence energytransfer tags, chemiluminescent compounds, chromophores, mass tags,electrophores, mononucleotides, oligonucleotides, or combinationsthereof.

Embodiment R71

The method of embodiment R70, wherein the detectable label of thecomplementary binding molecule comprises one or more fluorescence energytransfer tags.

Embodiment R72

The method of embodiment R71, wherein the complementary binding moleculefurther comprises one or more FRET cassettes.

Embodiment R73

The method of embodiment R72, wherein the FRET cassettes comprise one ormore dSpacer monomers.

Embodiment R74

The method of embodiment R73, wherein the complementary binding moleculehas the structure:

-   -   wherein T1 is a point of attachment for one or more fluorescent        energy donor or acceptor, and T2 is a point of attachment for        one or more of the complementary energy donor or acceptor to        that in Ti, wherein n is an integer between 1 and 20, and R        represents the point of attachment to the binder of the binding        molecule.

Embodiment R75

The method of embodiment R69, wherein the detectable label of thecomplementary binding molecule is one or more fluorophore.

Embodiment R76

The method of embodiment R75, wherein the fluorophore of the detectablelabel of the complementary binding molecule is selected from the groupconsisting of BodipyFL, R6G, ROX, Cy5, and Alexa488.

Embodiment R77

The method of any one of embodiments R69-R76, wherein the binder of thecomplementary binding molecule of each type of nucleotide analogue hasan anchor comprising:

-   -   a) a compound comprising streptavidin having the structure:

or

-   -   b) a compound comprising the structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to a detectable label.

Embodiment R78

The method of embodiment R77, wherein one type of nucleotide analoguehas an anchor having the structure:

-   -   wherein ω represents one or more atoms through which a covalent        connection is established to the cleavable t-butyldithiomethyl        moiety, and    -   the anchor orthogonally and rapidly reacts with a binder of a        complimentary binding molecule, wherein said binder comprises        streptavidin, and has the structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to a detectable label.    -   and thereby forms a conjugate having the structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to detectable label, and wherein ω is        one or more atoms through which a covalent connection is        established to the cleavable t-butyldithiomethyl moiety.

Embodiment R79

The method of embodiment R78, wherein the label is cleaved from theconjugate comprising the type of nucleotide analogue and bindingmolecule with citric acid/Na₂HPO₄.

Embodiment R80

The method of embodiment R78 or R79, wherein the type of nucleotideanalogue has the structure:

-   -   wherein base is one of adenine, guanine, thymine, cytosine,        uracil, or an analogue thereof.

Embodiment R81

The method of any one of embodiments R78-R80, wherein the complementarybinding molecule comprises streptavidin, and wherein the complementarybinding molecule has the structure:

Embodiment R82

The method of embodiment R77, wherein one type of nucleotide analoguehas an anchor moiety having the structure:

-   -   wherein ω represents one or more atoms through which a covalent        connection is established to the cleavable t-butyldithiomethyl        moiety, and    -   the anchor orthogonally and rapidly reacts with a binder of a        complimentary binding molecule, wherein said binder has the        structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to a detectable label, and    -   thereby forms a conjugate having the structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to detectable label, and wherein ω is        one or more atoms through which a covalent connection is        established to the cleavable t-butyldithiomethyl moiety.

Embodiment R83

The method of embodiment R82, wherein the label is cleaved from theconjugate comprising the type of nucleotide analogue and bindingmolecule with Na₂S₂O₄/H₂O.

Embodiment R84

The method of embodiment R82 or R83, wherein the type of nucleotideanalogue has the structure:

-   -   wherein base is one of adenine, guanine, thymine, cytosine,        uracil, or an analogue thereof.

Embodiment R85

The method of any one of embodiments R82-R84, wherein the complementarybinding molecule has the structure:

Embodiment R86

The method of embodiment R77, wherein one type of nucleotide analoguehas an anchor moiety having the structure

-   -   wherein ω represents one or more atoms through which a covalent        connection is established to the cleavable t-butyldithiomethyl        moiety, and    -   the anchor orthogonally and rapidly reacts with a binder of a        complimentary binding molecule, wherein said binder has the        structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to a detectable label, and    -   thereby forms a conjugate having the structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to detectable label, and wherein ω is        one or more atoms through which a covalent connection is        established to the cleavable t-butyldithiomethyl moiety.

Embodiment R87

The method of embodiment R86, wherein the type of nucleotide analoguehas the structure:

-   -   wherein base is one of adenine, guanine, thymine, cytosine,        uracil, or an analogue thereof.

Embodiment R88

The method of embodiment R86 or R87, wherein the complementary bindingmolecule has the structure:

Embodiment R89

The method of embodiment R77, wherein one type of nucleotide analoguehas an anchor having the structure:

-   -   wherein ω represents one or more atoms through which a covalent        connection is established to the cleavable t-butyldithiomethyl        moiety, and    -   the anchor orthogonally and rapidly reacts with a binder of a        complimentary binding molecule, wherein said binder has the        structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to a detectable label, and    -   thereby forms a conjugate having the structure:

-   -   wherein α is one or more atoms through which a covalent        connection is established to detectable label, and wherein ω is        one or more atoms through which a covalent connection is        established to the cleavable t-butyldithiomethyl moiety.

Embodiment R90

The method of embodiment R89, wherein the label is cleaved from theconjugate comprising the type of nucleotide analogue and bindingmolecule with citric acid/Na₂HPO₄.

Embodiment R91

The method of embodiment R89 or R90, wherein the type of nucleotideanalogue has the structure:

-   -   wherein base is one of adenine, guanine, thymine, cytosine,        uracil, or a derivative thereof.

Embodiment R92

The method of any one of embodiments R89-R91, wherein the complementarybinding molecule has the structure:

Embodiment R93

The method of any one of embodiments R58-R92, wherein the cleavablet-butyldithiomethyl moiety, has the structure:

-   -   wherein α represents the point of connection to the 3′-oxygen.

Embodiment R94

The method of embodiment R93, wherein the cleavable t-butyldithiomethylmoiety has the structure:

-   -   wherein α represents the point of connection to the 3′-oxygen;        and wherein n is an integer which may be 1, 2, 3, 4, or 5.

Embodiment R95

The method of any one of embodiments R65-R66, or R68, wherein the fourthtype of nucleotide analogue has the structure:

Embodiment R96

The method of any one of embodiments R68-R95, wherein the cleavablet-butyldithiomethyl moiety may be cleaved by a water soluble phosphine,thereby resulting in a 3′-OH.

Embodiment R97

The method of embodiment R96, wherein the water soluble phosphine istris-(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine(THP).

Embodiment R98

A method for determining the identity of a nucleotide at a predeterminedposition in a nucleic acid of interest, comprising:

-   -   a) providing        -   1) the nucleic acid of interest,        -   2) a nucleic acid polymerase,        -   3) a primer capable of hybridizing to said nucleic acid            immediately 3′ of such predetermined position,        -   4) four different nucleotide analogues of embodiment 9, each            of which consists of one of adenine or an analogue of            adenine, guanine or an analogue of guanine, cytosine or an            analogue of cytosine, thiamine or an analogue of thiamine,            and a unique detectable label;    -   b) incorporating one of said nucleotide analogues onto the end        of said primer to form an extension strand;    -   c) detecting the unique detectable label of the incorporated        nucleotide analogue so as to thereby identify the incorporated        nucleotide analogue on the end of said extension strand; and    -   d) based on the identity of the incorporated nucleotide,        determining the identity of the nucleotide at the predetermined        position.

Embodiment R99

The method of embodiment R98 further comprising, treating the extensionstrand of step (b) so as to cleave the t-butyldithiomethyl moiety boundto the 3′-oxygen of the sugar and so as to produce a 3′-OH on the sugarand for producing an extension, remove the label from the extensionstrand to which another nucleotide analogue may be added.

Embodiment R100

The method of embodiment R99, wherein treatment comprises contacting theextension strand with tris-(2-carboxyethyl)phosphine (TCEP) ortris(hydroxypropyl)phosphine (THP).

Embodiment R101

The method of any one of embodiments R98-R100, wherein each nucleotideanalogue is a nucleotide triphosphate, a nucleotide tetraphosphate, anucleotide pentaphosphate, or a nucleotide hexaphosphate.

Embodiment R102

The method of any one of embodiments R98-R101, wherein the nucleotideanalogues comprise a deoxyribose.

Embodiment R103

The method embodiment R102, wherein the polymerase is a DNA polymeraseand the nucleic acid is DNA.

Embodiment R104

The method of any one of embodiments R98-R101, wherein the nucleotideanalogues comprise a ribose.

Embodiment R105

The method of embodiment R104, wherein the polymerase is a reversetranscriptase and the nucleic acid is RNA.

Embodiment R106

The method of embodiment R102, wherein the polymerase is a DNA-based RNApolymerase and the nucleic acid is DNA.

Embodiment R107

The method of embodiment R104, wherein the polymerase is an RNA-basedRNA polymerase and the nucleic acid is RNA.

Embodiment R108

The method of any one of embodiments R98-R108, wherein thet-Butyldithiomethyl linker has the structure:

-   -   wherein α represents the point of connection to the 3′-oxygen;    -   wherein R represents one or more atoms through which a covalent        connection is established to the detectable label; and    -   wherein Label is the detectable label.

Embodiment R109

The method of any one of embodiments R98-R108, wherein thet-Butyldithiomethyl linker has the structure:

-   -   wherein α represents the point of connection to the 3′-oxygen;    -   wherein n is 1, 2, 3, 4, or 5; and    -   wherein R′ represents one or more atoms through which a covalent        connection is established to the detectable label.

Embodiment R110

The method of any one of embodiments R98-R109, wherein the detectablelabel is selected from the group consisting of a dye, a fluorophore, acombinatorial fluorescence energy transfer tag, a chemiluminescentcompound, a chromophore, a mass tag, an electrophore, a mononucleotide,an oligonucleotide, or a combination thereof.

Embodiment R111

The method of embodiment R110, wherein the detectable label is afluorophore.

Embodiment R112

The method of embodiment R112, wherein the fluorophore is selected fromthe group consisting of BodipyFL, R6G, ROX, Cy5, and Alexa488.

Embodiment R113

The method of any one of embodiments R98-R112, wherein each nucleotideanalog is selected from the group consisting of3′-O-Alexa488-t-Butyldithiomethyl-dCTP,3′-O-Cy5-t-Butyldithiomethyl-dGTP, 3-O-Rox-t-Butyldithiomethyl-dATP,3′-O-RG6-t-Butyldithiomethyl-dTTP,3′-O-Alexa488-PEG₄-t-Butyldithiomethyl-dCTP,3′-O-RG6-PEG₄-t-Butyldithiomethyl-dTTP,3′-O-Rox-PEG₄-t-Butyldithiomethyl-dATP,3′-O-Cy5-PEG₄-t-Butyldithiomethyl-dGTP.

Embodiment R114

The method of any one of embodiments R98-R110, wherein the structure ofeach labeled nucleotide analog is selected from:

Embodiment R115

The method of any one of embodiments R98-R114, wherein the nucleic acidof interest is immobilized on a solid support.

Embodiment R116

The method of embodiment R115, wherein the nucleic acid of interest isimmobilized on the solid support via an azido linkage, an alkynyllinkage, a 1,3-dipolar cycloaddition linkage, or a biotin-streptavidininteraction.

Embodiment R117

The method of any one of embodiments R115-R116, wherein the solidsupport is in the form of a chip, a bead, a well, a capillary tube, or aslide.

Embodiment R118

The method of any of embodiments R115-R117, wherein the solid supportcomprises gold, quartz, silica, or a plastic.

Embodiment R119

The method of any of embodiments R115-R117, wherein the solid support isporous.

Embodiment R120

A method of sequencing a nucleic acid of interest which comprisesrepeatedly determining the identity of each nucleotide present in thenucleic acid of interest according to the method of any one ofembodiments R98-R119.

Embodiment R121

A method of simultaneously sequencing a plurality of different nucleicacids of interest which comprises simultaneously sequencing each suchnucleic acid according to the method of embodiment R120.

Embodiments for 3′ Anchor Tags for SBS Embodiment J1

A method for determining the nucleotide sequence of a single-strandedDNA including:

contacting the single-stranded DNA, wherein the single-stranded DNA isbound to a polymerase which is in turn attached to a membrane-embeddednanopore in an electrolyte solution, wherein the single-stranded DNA hasa primer hybridized to a portion thereof, and determining the sequenceof the single stranded DNA template, following the steps of

-   -   (a) addition of four nucleotides including 3′-O-cleavable        linkers (DTM) attached with anchor moieties. The appropriate        nucleotide analogue complementary to the nucleotide residue of        the single-stranded DNA (template) which is immediately 5′ to a        nucleotide residue of the single-stranded DNA will be        incorporated by DNA polymerase at the 3′ terminal nucleotide        residue of the primer, so as to form a DNA extension product.        Only a single 3′-O-anchor-cleavable linker (DTM) nucleotide will        add to the primer due to the 3′-O-being blocked by a cleavable        linker and anchor moiety, preventing further incorporation in        this step;    -   (b) addition to the extended primer of 4 different nanopore tags        attached with different binding molecules corresponding to the 4        anchors; the appropriate binding molecule with tag will either        covalently bind or complex with the 3′-O-anchor nucleotide        incorporated in step (a);    -   (c) application of a voltage across the membrane and measuring        an electronic (ionic current) change across the nanopore        resulting from the tag attached thereto generated in step (b)        translocating through the nanopore, wherein the electronic        change is different for each different type of tag, thereby        identifying the nucleotide residue in the single-stranded        template DNA, which is complementary to the incorporated tagged        nucleotide;    -   (d) cleavage of the 3′-O-cleavable linker-attached tag by        treatment with an appropriate cleaving agent, thus generating a        free 3′-OH ready for the next extension reaction.    -   (e) Iteratively performing steps (a)-(d) for each nucleotide        residue of the single-stranded DNA being sequenced, wherein in        each iteration of step (a) the 3′-O-cleavable anchor nucleotide        is incorporated into the DNA extension product resulting from        the previous iteration of step (d) if it is complementary to the        nucleotide residue of the single-stranded (template) DNA which        is immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the DNA        extension product, thereby determining the nucleotide sequence        of the single-stranded DNA.

Embodiment J2

The method of Embodiment J1 wherein each of the at least four3′-O-Anchor-Cleavable Linker nucleotides includes a triphosphate or apolyphosphate, a base which is adenine, guanine, cytosine, thymine, oruracil, or a derivative of each thereof, and an anchor moleculecovalently coupled to the 3′-O-position of the nucleotide sugar moietyincluding a cleavable linker at the 3′-O-position;

Embodiment J3

The method of Embodiment J1 wherein the cleavable linker is dithiomethyl(SS(DTM)), Allyl, Azo, or 2-Nitrobenzyl-based linkers.

Embodiment J4

The method of Embodiment J1 wherein the cleavable linker is cleaved byDTT, THP, TCEP, Pd(0), sodium dithionite, or UV light of approximately340 nm.

Embodiment J5

The method of Embodiment J1 wherein the anchor moiety includes biotin,azide, trans-cyclooctene (TCO), phenylboronic acid (PBA), quadricyclane,or norbornene.

Embodiment J6

The method of Embodiment J1 wherein the anchor binding partner moleculeincludes streptavidin, dibenzylcyclooctene (DBCO), tetrazine,salicylhydroxamic acid (SHA), bis(dithiobenzil)nickel(II) compounds,nitrile oxide containing compounds

Embodiment J7

The method of Embodiment J1 wherein the nanopore tag is anoligonucleotide, peptide, PEG, carbohydrate or a combination thereof.

Embodiment J8

A method for determining the nucleotide sequence of a single-strandedDNA including:

contacting the single-stranded DNA template, wherein the single-strandDNA to be sequenced hybridizes to the primer, wherein thesingle-stranded primer is conjugated to a membrane-embedded nanopore inan electrolyte solution, and determining the sequence of the singlestranded DNA template, following the steps of

-   -   (a) addition of polymerase and four nucleotides including        3′-O-cleavable linkers (DTM) attached with anchor moieties. The        appropriate nucleotide analogue complementary to the nucleotide        residue of the single-stranded DNA (template) which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA will be incorporated by DNA polymerase at the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product. Only a single 3′-O-anchor-cleavable linker (DTM)        nucleotide will add to the primer due to the 3′-O-being blocked        by a cleavable linker and anchor moiety, preventing further        incorporation in this step;    -   (b) addition to the extended primer of 4 different nanopore tags        attached with different binding molecules corresponding to the 4        anchors; the appropriate binding molecule with tag will either        covalently bind or complex with the 3′-O-anchor nucleotide        incorporated in step (a);    -   (c) application of a voltage across the membrane and measuring        an electronic (ionic current) change across the nanopore        resulting from the tag attached thereto generated in step (b)        translocating through the nanopore, wherein the electronic        change is different for each different type of tag, thereby        identifying the nucleotide residue in the single-stranded        template DNA, which is complementary to the incorporated tagged        nucleotide;    -   (d) cleavage of the 3′-O-cleavable linker-attached tag by        treatment with an appropriate cleaving agent, thus generating a        free 3′-OH ready for the next extension reaction.    -   (e) Iteratively performing steps (a)-(d) for each nucleotide        residue of the single-stranded DNA being sequenced, wherein in        each iteration of step (a) the 3′-O-cleavable anchor nucleotide        is incorporated into the DNA extension product resulting from        the previous iteration of step (d) if it is complementary to the        nucleotide residue of the single-stranded (template) DNA which        is immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the DNA        extension product, thereby determining the nucleotide sequence        of the single-stranded DNA.

Embodiment J9

The method of Embodiment J8 wherein each of the at least four3′-O-Anchor-Cleavable Linker nucleotides includes a triphosphate or apolyphosphate, a base which is adenine, guanine, cytosine, thymine, oruracil, or a derivative of each thereof, and an anchor moleculecovalently coupled to the 3′-O-position of the nucleotide sugar moietyincluding a cleavable linker at the 3-O-position;

Embodiment J10

The method of Embodiment J8 wherein the cleavable linker is adithiomethyl (SS(DTM)), Allyl, Azo. or 2-Nitrobenzyl-based linkers.

Embodiment J11

The method of Embodiment J8 wherein the cleavable linker is cleaved byDTT, THP, TCEP, Pd(0), sodium dithionite, or UV light of approximately340 nm.

Embodiment J12

The method of Embodiment J8 wherein the anchor moiety includes biotin,azide, trans-cyclooctene (TCO), phenylboronic acid (PBA), quadricyclane,or norbornene.

Embodiment J13

The method of Embodiment J8 wherein the anchor binding partner moleculeincludes streptavidin, dibenzylcyclooctene (DBCO), tetrazine,salicylhydroxamic acid (SHA), bis(dithiobenzil)nickel(II) compounds,nitrile oxide containing compounds

Embodiment J14

The method of Embodiment J8 wherein the nanopore tag is anoligonucleotide, peptide, PEG, carbohydrate or a combination thereof.

Embodiment J15

The methods of Embodiments J1 and J8, wherein 4 nucleotide analogsattached to a cleavable linker including 4 different anchor moleculesare added, followed by the addition of 4 comparable anchor bindingmolecules attached to 4 different nanopore tags.

Embodiment J16

The methods of Embodiments J1 and J8, wherein 3 nucleotide analogsattached to a cleavable linker including 3 different anchor moleculesand 1 nucleotide analog attached to a cleavable linker lacking an anchormolecule are added, followed by the addition of 3 comparable anchorbinding molecules attached to 3 different nanopore tags:

Embodiment J17

The methods of Embodiments J1 and J8, wherein each of the 4 nucleotideshas a different combination of anchor and cleavable linker, and 2different binding molecules attached to 2 different nanopore tags.

Embodiment J18

The method of Embodiment J17, wherein one of the nucleotides is an azidoanchor and a SS(DTM) cleavable linker, the second nucleotide is an TCOanchor and a SS(DTM) cleavable linker, the third nucleotide is an azidoanchor and a 2-nitrobenzyl cleavable linker, and the fourth nucleotideis a TCO anchor and a 2-nitrobenzyl cleavable linker.

Embodiment J19

The method of Embodiment J17, wherein one of the nucleotides is an azidoanchor and a SS(DTM) cleavable linker, the second nucleotide is an TCOanchor and a SS(DTM) cleavable linker, the third nucleotide is an azidoanchor and a Azo cleavable linker, and the fourth nucleotide is a TCOanchor and a Azo cleavable linker.

Embodiment J20

The method of Embodiment J17, wherein one of the nucleotides is an azidoanchor and a SS(DTM) cleavable linker, the second nucleotide is an TCOanchor and a SS(DTM) cleavable linker, the third nucleotide is an azidoanchor and an allyl cleavable linker, and the fourth nucleotide is a TCOanchor and an allyl cleavable linker.

Embodiment J21

The methods of Embodiments J17-J20, wherein the anchor binding moleculesinclude DBCO attached to one nanopore tag and tetrazine attached to adifferent nanopore tag.

Embodiment J22

The methods of Embodiments J17-J20, wherein the nanopore tag is anoligonucleotide, PEG, peptide, or carbohydrate chain.

Embodiment J23

The methods of Embodiments J1 and J8, wherein the four nucleotidesinclude 3′-O-Anchor-Cleavable Linker (DTM) nucleotides;

Embodiment J24

The methods of Embodiments J1 and J8, wherein the anchor moiety attachedto the 3′-O-DTM nucleotides is selected from azide, trans-cyclooctene(TCO), PBA, and quadricyclane (QC).

Embodiment J25

The methods of Embodiments J1 and J8, wherein the anchor bindingmolecule attached to the nanopore tags is selected from DBCO, tetrazine.SHA, and Ni-bis(dithioline) compounds.

Embodiment J26

The methods of Embodiments J1 and J8, wherein the cleavable linker (DTM)is cleaved by DTT, TCEP or THP.

For Mixture of Nucleotide Analogs with Dyes on Base, and Label (Dye orAnchor) on 3′ Position (FIGS. 70-76)

Embodiment K1

A method of sequencing nucleic acid, including: a) extending a primingstrand of DNA by incorporating a labeled nucleotide into the primingstrand; and b) identifying the labeled nucleotide, so as to sequence thenucleic acid.

Embodiment K2

The method of embodiment K1, wherein the labeled nucleotide has thelabel linked to the base and a cleavable blocking group on the3′-hydroxyl group.

Embodiment K3

The method of embodiment K1, wherein the labeled nucleotide has thelabel linked to the 3′ OH through a cleavable linker.

Embodiment K4

The method of embodiment K2, wherein the label is attached to the basevia a cleavable linker.

Embodiment K5

The method of embodiments K2 to K4, wherein the chemically cleavablelinker is dithiomethyl SS(DTM), Azo, allyl or 2-nitrobenzyl.

Embodiment K6

The method of embodiments K2 to K3, wherein the 3′ OH blocking group isSS(DTM), azidomethyl, Azo, allyl or 2-nitrobenzyl.

Embodiment K7

The method of embodiment K1, wherein the nucleotide analog includes adeazapurine base.

Embodiment K8

A method of sequencing nucleic acid including: a) providing a nucleicacid template hybridized to a primer; b) extending the primer hybridizedto the nucleic acid template with a labeled nucleotide or nucleotideanalog, wherein the labeled nucleotide or nucleotide analog includesnucleotide analogs with a label linked to the base and a blocking groupon the 3′-hydroxyl group, and nucleotides or nucleotide analogs with acleavable label blocking the 3′ OH; and c) identifying the labelednucleotide, so as to sequence the nucleic acid.

Embodiment K9

The method of embodiment 8, wherein the labeled nucleotide or nucleotideanalog includes nucleotide analogs with a label linked to the base and ablocking group on the 3′-hydroxyl group, and nucleotides or nucleotideanalogs with a cleavable label blocking the 3′ OH.

Embodiment K10

The method of embodiment K9, wherein the label is attached to the baseor blocking the 3′ OH group with a cleavable linker.

Embodiment K11

The method of embodiment K10, wherein the cleavable linker is achemically cleavable linkers.

Embodiment K12

The method of embodiment K10, wherein the chemically cleavable linker isdithiomethyl SS(DTM), Azo, allyl or 2-nitrobenzyl.

Embodiment K13

The method of embodiment K8, wherein the nucleotide analog includes adeazapurine base.

Embodiment K14

A method of simultaneously sequencing a plurality of different nucleicacids, including: a) extending a plurality of priming DNA strandshybridized to template DNAs, each of which includes one of the primingDNA strands, by incorporating a labeled nucleotide; and b) identifyingeach labeled nucleotide, so as to simultaneously sequence the pluralityof different nucleic acids.

Embodiment K15

The method of embodiment K14, wherein the labeled nucleotide ornucleotide analog includes nucleotide analogs with a label linked to thebase and a blocking group on the 3′-hydroxyl group, and nucleotides ornucleotide analogs with a cleavable label blocking the 3′ OH.

Embodiment K16

The method of embodiment K15, wherein the label is attached to the basevia a cleavable linker.

Embodiment K17

The method of embodiment K14, wherein the 3′ OH blocking group isattached to the deoxyribose via a cleavable linker.

Embodiment K18

The method of embodiment K14, wherein the cleavable linker is chemicallycleavable linkers.

Embodiment K19

The method of embodiment K18, wherein the chemically cleavable linker isdithiomethyl SS(DTM), Azo, allyl or 2-nitrobenzyl.

Embodiment K20

The method of embodiment K14, wherein the 3′ OH blocking group isSS(DTM), azidomethyl, Azo, allyl or 2-nitrobenzyl.

Embodiment K21

The method of embodiment K14, wherein the nucleotide analogue includes adeazapurine base.

EXAMPLES

Among various new DNA sequencing methods, sequencing by synthesis (SBS)is the leading method for realizing the goal of the $1,000 genome.Currently, the widely used high-throughput SBS technology (Bentley(2008)) determines DNA sequences during the polymerase reaction usingcleavable fluorescently labeled nucleotide reversible terminator (NRT)sequencing chemistry that have been previously developed (Ju et al.(2003); Ju et al. (2006)). These cleavable fluorescent NRTs weredesigned such that each of the four nucleotides (A, C, G, T) is modifiedby attaching a unique cleavable fluorophore to the specific location ofthe base and capping the 3′-OH group with a small reversibly-blockingmoiety so they are still recognized by DNA polymerase as substrates.Thus, the cleavable fluorescent NRTs involve two modifications inseparate locations of the nucleotide (Ju et al. (2003); Ju et al.(2006)); Bentley et al. 2008): (1) a fluorescent dye to serve as areporter group on the base; and (2) a small chemical moiety to cap the3′-OH group to temporarily terminate the polymerase reaction afternucleotide incorporation for sequence determination. After incorporationand signal detection, the fluorophore is cleaved and the 3′-OH cappingmoiety removed to resume the polymerase reaction in the next cycle.These cleavable fluorescent NRTs have proved to be good substrates forre-engineered polymerases and have been used extensively in nextgeneration DNA sequencing systems (Ju (2006); Bentley (2008)). Moreover,they enable accurate determination of homopolymer sequences, since onlyone base is identified in each cycle.

Fluorescence-based methods have many advantages in terms of detectionsensitivity. However, because of the large size of the fluorophores,specific polymerase and reaction conditions need to be optimized forsequencing reactions. In addition, the current cleavable fluorescentNRTs used in SBS leave a modified group on the base of the growing DNAstrand after cleavage of the fluorophore, limiting sequencing readlength.

As an alternative to fluorescence-based DNA SBS, an approach has beenpreviously reported, which uses an azido moiety (N₃) that has anintense, narrow and unique Raman shift at 2125 cm⁻¹, where virtually allbiological molecules are transparent, as a label for SBS (Palla (2014)).The azido label is part of the moiety that also serves as a reversibleblocking group for the 3′-OH group of the nucleotides. The extended DNAstrand from these nucleotides is identical to natural DNA. This isunlike many, current SBS approaches, which require the use of modifiednucleotides that leave short remnants of the linkers after cleavage ofthe fluorescent tags (Ju (2006); Bentley (2008); Harris (2008)); asthese remnants build up in the extended DNA chains, they areincreasingly likely to alter DNA structure and impede further nucleotideincorporation by polymerase.

Fluorescent NRTs with the following blocking groups at the 3′-OH havebeen reported: 3′-O-allyl-dNTP(Bentley (2008)), 3′-O-azidomethyl-dNTPs(Wu (2007); Guo (2008): Bentley (2008)), 3′-O-NH₂-dNTPs (Hunter (2010)),and 3′-O-cyanoethyl-dNTPs (Knapp (2011)), which can be cleaved by Pd(0),tris(2-carboxyethyl)phosphine (TCEP), dilute nitrous acid and fluoride,respectively, to generate the free 3′-OH group.

Various modifications based on 3′-O-alkyldithiomethyl (3′-O-DTM) for thenucleosides (Kwiatkowski (2007); Muller (2011); Semenyuk (2010)) werereported for the synthesis of oligonucleotides. The stability andreductive cleavage leading to hydroxyl production from the O-DTM grouphave been established (Kwiatkowski (2007); Muller (2011); Semenyuk(2010)), but their utility in DNA sequencing applications has not beenreported. This is due to the fact that nucleotide analogs with a largefluorescent dye blocking the 3′-OH group were not reported to beincorporated by DNA polymerase in template-directed DNA synthesis.

DNA sequencing is a fundamental tool in biological and medical research;it is an essential technology for the paradigm of personalized precisionmedicine. Among various new DNA sequencing methods, sequencing bysynthesis (SBS) is the leading method for realizing the goal of the$1,000 genome. SBS determines DNA sequences during the polymerasereaction. Currently, the widely used high-throughput SBS technology(Bentley (2008)) determines DNA sequences during the polymerase reactionusing cleavable fluorescently labeled nucleotide reversible terminator(NRT) sequencing chemistry that has been previously developed (Ju et al.(2003); Ju et al. (2006)). These cleavable fluorescent NRTs weredesigned based on the rationale that each of the four nucleotides (A, C,G, T) is modified by attaching a unique cleavable fluorophore to thespecific location of the base and capping the 3′-OH group with a smallreversible-blocking moiety so they are still recognized by DNApolymerase as substrates. Thus, the cleavable fluorescent NRTs involvetwo modifications in separate locations of the nucleotide (Ju et al.(2003); Ju et al. (2006); Bentley et al. (2008)): (1) a fluorescent dyeto serve as a reporter group on the base; and (2) a small chemicalmoiety to cap the 3′-OH group to temporarily terminate the polymerasereaction after nucleotide incorporation for sequence determination.After nucleotide incorporation and signal detection to identify theincorporated nucleotide, the fluorophore is cleaved and the 3′-OHcapping moiety is removed, enabling the polymerase reaction to resume inthe next cycle. These cleavable fluorescent NRTs have proved to be goodsubstrates for re-engineered polymerases and have been used extensivelyin next generation DNA sequencing systems (Ju (2006); Bentley (2008)).Moreover, they enable accurate determination of homopolymer sequences,since only one base is identified in each cycle.

It is known that nucleotides modified with bulky groups such as energytransfer dyes at the 5-position of the pyrimidines (T and C) and the7-position of purines (G and A) are still recognizable by engineered DNApolymerase as substrates (Rosenblum (1997); Zhu (1994). The ternarycomplexes of a rat DNA polymerase, a DNA template-primer, anddideoxycytidine triphosphate have been determined (Pelletier (1994)),which supports these findings. Thus, if a unique fluorescent dye islinked to the 5-position of the pyrimidines (T and C) and the 7-positionof purines (G and A) via a cleavable linker, and a small chemical moietyis used to cap the 3′-OH group, the resulting nucleotide analoguesshould incorporate into the growing DNA strand as terminators. Based onthis rationale, an SBS approach using cleavable fluorescent nucleotideanalogues as reversible terminators to sequence surface-immobilized DNAwas developed (Ju (2003). Ruparel (2005); Marguiles (2005); Ju (2006);Wu (2007); Guo (2008)). In this approach, the nucleotides are modifiedat two specific locations so that they are still recognized by DNApolymerase as substrates: (1) a different fluorophore with a distinctfluorescent emission is attached to the specific location of each of thefour bases through a cleavable linker and (ti) the 3′-OH group is cappedby a small chemically reversible moiety. DNA polymerase onlyincorporates a single nucleotide analogue complementary to the base on aDNA template covalently linked to a surface. After incorporation, theunique fluorescence emission is detected to identify the incorporatednucleotide. The fluorophore is subsequently removed and 3′-OH group ischemically regenerated, which allows the next cycle of the polymerasereaction to occur. Because a high density of different DNA templates canbe spotted on the large surface of a DNA chip, each cycle can identifymany bases in parallel, allowing the simultaneous sequencing of a largenumber of DNA molecules.

Fluorescence-based methods have many advantages in terms of detectionsensitivity. However, because of the large size of the fluorophores,specific polymerase and reaction conditions need to be optimized forsequencing reactions. An additional disadvantage of the abovementionedSBS approach is the production of a small molecular “scar” (often apropargylamine or a modified propargylamino moiety) at the nucleotidebase after cleavage of the fluorescent dye from the incorporatednucleotide in the polymerase reaction. The growing DNA chain accumulatesthese scars through each successive round of SBS. At some point, theresidual scars may be significant enough to interfere with the DNAdouble helix structure, thereby negatively affecting DNA polymeraserecognition and consequently limiting the read length.

Due to the desirability of increasing SBS read-length, SBS schemes havebeen explored in which the “reporter” dye is attached directly to the3′-OH group of the nucleotide analogues via a cleavable linker that willallow scarless SBS to take place. In such a scarless SBS process, afternucleotide incorporation and imaging of the reporter moiety on theincorporated 3′-O modified nucleotide for sequence determination, thecleavage of the linker would generate a free 3′-OH group on the growingDNA strand for subsequent extension reactions. Earlier work was focusedon designing and synthesizing a cleavable chemical moiety that waslinked to a fluorescent dye to cap the 3′-OH group of the nucleotidesusing 3′-O-ester linkage (Cheeseman (1994); Canard (1994)). However,these nucleotide analogues were largely unsuccessful in SBS schemesbecause DNA polymerase had difficulty accepting these nucleotideanalogues as a substrate. Aiming to create a high-throughput DNAsequencing platform, other groups also pursued modified nucleotides witha reversible 3′-O fluorescent dye (Welch (1999); Metzker (2005); Lu(2006)). Accumulated research efforts indicated that the major challengefor this approach is that DNA polymerase has difficulty accepting 3-O0bulky-dye-modified nucleotides as substrates, because the 3′ position onthe deoxyribose of the nucleotides is very close to the amino acidresidues in the active site of the DNA polymerase while in the ternarycomplex formed by the polymerase with the complementary nucleotide andthe primed template. Recently, Kim et. al. reported 3′-O-fluorescentlymodified nucleotides using an allyl linker to attach small fluorescentdyes (coumarin, Pacific Blue and BodipyFL), which are reasonably goodsubstrates for a Therminator II DNA polymerase. However, nucleotidesmodified with bulky dyes or highly charged dyes (such as Alexa 488)using the same linker are not suitable substrates for DNA polymerase(Kim (2010); Kim (2014).

To enable long read-length in SBS, it is essential for the cleavablelinker to be stable during the sequencing reactions, with a minimalnumber of cycles and to leave no scars on the base after the cleavagereaction. Nucleotide analogues with reporter molecules attached to the3′-O via a cleavable linker are ideal for this purpose; such modifiednucleotides would generate naturally elongated DNA during the DNAsynthesis. However the major challenge is designing and synthesizingthis type of modified nucleotide analogue that is accepted by DNApolymerase as a substrate. The NRTs with the following blocking groupsat the 3′-OH of the nucleotide have been reported and shown to be goodsubstrates for DNA polymerases: 3′-O-(2-nitrobenzyl)-dNTPs (Wu (2007)),3′-O-allyl-dNTPs (Ju (2003); Ju (2006)), 3′-O-azidomethyl-dNTPs (Guo(2008); Bentley (2008)), 3′-O-NH₂ (Hutter (2010)), and 3′-O-cyanoethyl(Diana (2011)). The 3′ blocking moieties in all these molecules can bereadily cleaved to regenerate the 3′-OH group. This combined researchindicates that 3′-O-NRTs with a small chemical moiety attached to the3′-OH group are good substrates for DNA polymerases and are ideal forconducting DNA SBS. Various 3′-O-t-butyldithiomethyl (3′-O-DTM) basedmodifications on nucleosides (Kwiatkowski (2007); Muller (2011);Semenyuk (2006)) have been reported for the synthesis ofoligonucleotides. The reductive cleavage leading to hydroxyl productionfrom O-DTM group has been well established (Kwiatkowski (2007); Muller(2011); Semenyuk (2006)), but the utility of these types of moleculeswith the 3′-O-DTM modification in DNA SBS applications has not beenreported. Accordingly, there is a need for the use in scarless SBS, andsynthesis of, 3′-O modified nucleotides and nucleosides that areeffectively recognized as substrates by DNA polymerases, are efficientlyand accurately incorporated into growing DNA chains during SBS, have a3′-O blocking group that is cleavable under mild conditions whereincleavage results in a 3′-OH, and permit long SBS read-lengths.

Example 1: Synthesis and Characterization of 3′-O-Dye-DTM-dNTPs

Fluorescence-based DNA sequencing-by-synthesis methods have manyadvantages in terms of detection sensitivity. However, because of thelarge size of the fluorophores, specific polymerase and reactionconditions need to be optimized for sequencing reactions. In addition,the current cleavable fluorescent nucleotide reversible terminators usedin SBS leave a modified group, or scar, on the base of the growing DNAstrand after cleavage of the fluorophore, which in turn limits readlength.

Fluorescent NRTs with the following blocking groups at the 3′-OH havebeen reported: 3′-O-allyl-dNTP(Bentley (2008)), 3′-O-azidomethyl-dNTPs(Wu (2007); Guo (2008); Bentley (2008)), 3′-O-NH₂-dNTPs (Hunter (2010)),and 3′-O-cyanoethyl-dNTPs (Knapp (2011)), which can be cleaved by Pd(0),tris(2-carboxyethyl)phosphine (TCEP), dilute nitrous acid and fluoride,respectively, to generate the free 3′-OH group.

Various modifications based on 3′-O-alkyldithiomethyl (3′-O-DTM) for thenucleosides (Kwiatkowski (2007); Muller (2011); Semenyuk (2010)) havebeen reported for the synthesis of oligonucleotides. The stability andreductive cleavage leading to hydroxyl production from the O-DTM grouphas also been established (Kwiatkowski (2007); Muller (2011); Semenyuk(2010)), but their utility in DNA sequencing applications has not beenreported. This is because nucleotide analogs with a large fluorescentdye blocking the 3′-OH group were reported to not be incorporated by DNApolymerase in template-directed DNA synthesis.

By the unique chemical design of the cleavable linker attached to afluorescent dye to block the 3′-OH group of the nucleotide, coupled withspecific polymerase reaction conditions, it is herein disclosed that themodified 3′-O-dithiomethyl (3′-O-DTM) is a successful reversible linkagegroup for attaching a fluorescent dye reporter to block the 3′-OH groupof the nucleotide for DNA SBS. To this end, herein disclosed are novel3′ reversibly labeled nucleotides as traceless reversible terminators,which were designed and synthesized for DNA SBS. In these novelnucleotide analogs, only the 3′-OH group of the nucleotide is reversiblyblocked with a DTM linker, which is attached to the fluorescent label,thus realizing the dual function of the 3′-O-modification of thenucleotide, serving as both the reversible terminator function and thecleavable fluorescence reporter.

It is further disclosed herein, that in SBS cycles, such3′-O-Dye-DTM-dNTPs are well recognized by the DNA polymerase,Therminator (9° N DNA polymerase variant), as substrates andincorporated into the growing DNA strand. After determining the identityof the incorporated nucleotide by its fluorescent signal, TCEP orTris(3-hydroxypropyl)phosphine (THP) treatment cleaves the disulfidebond in the DMT moiety leading to both the removal of the fluorescencereporter and the regeneration of the 3′-OH group to allow for continuoussequencing. After each incorporation and cleavage, an extended naturalDNA strand is produced to allow for the seamless incorporation ofincoming complementary 3′-O-Dye-DTM-dNTPs during SBS.

There are surprising advantages to using 3′-O-Dye-DTM-dNTPs for SBS. Asdisclosed herein, consecutive polymerase extension reaction using3′-O-Dye-DTM-dNTPs with a synthetic template and primer have beencarried out. After single base extension and cleavage of the DTM moietyand the removal of dye from the 3′-O of the DNA extension product, theresulting primer extension product can be further extended with anadditional 3′-O-Dye-DTM-dNTP, leading to a high-yield incorporation withaccurate sequence determination. Because these 3′-O-Dye-DTM-dNTPs do notrequire the attachment of fluorescent labels on the base, theirsynthesis is simpler and therefore more cost effective. In addition, theextended DNA strand is identical to natural DNA. The use of3′-O-Dye-DTM-dNTPs will lead to very long, accurate read lengths forSBS.

The Synthesis of 3′-O-Bodipy-DTM-dTTP and 3′-O-Bodipy-PEG₄-DTM-dTTP

3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl thymidine (T2): To asolution of the 5′-O-tert-Butyldimethylsilyl thymidine (T1, 1.07 g, 3mmol) in DMSO (10 mL) with stirring was added acetic acid (2.6 mL) andacetic anhydride (8.6 mL). The reaction mixture was stirred at roomtemperature until the reaction was complete (48 h), which was monitoredby TLC. Then the mixture was added slowly to the solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the residue of the desired compound was purified by silicagel column chromatography (ethyl acetate/hexane: 1/2) to give pureproduct T2 (0.97 g, 74%). ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (s, 1H), 7.48(s, 1H), 6.28 (m, 1H), 4.62 (m, 2H), 4.46 (m, 1H), 4.10 (m, 1H),3.78-3.90 (m, 2H), 2.39 (m, 1H), 2.14 (s, 3H), 1.97 (m, 1H), 1.92 (s,3H), 0.93 (s, 9H), 0.13 (s, 3H); HRMS (Fab⁺) calc'd for C₁₈H₃₃N₂O₅SSi[(M+H)+]: 417.1879, found: 417.1890.

Compound T3:

3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl thymidine (T2, 625mg, 1.50 mmol) was dissolved in anhydrous dichloromethane (20 mL),followed by addition of triethylamine (0.3 mL) and molecular sieves (3Å, 2 g). The mixture was cooled in an ice-bath after stirring at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.12mL, 1.50 mmol) in anhydrous dichloromethane (3 mL) was added dropwiseduring 2 minutes. The ice-bath was removed and the reaction mixture wasstirred further for 0.5 hour. Then potassium p-toluenethiosulfonate(0.61 g, 2.25 mmol) in anhydrous DMF (3 mL) was added to the mixture.Stirring was continued at room temperature for additional 1 hourfollowed by addition of2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide (403 mg, 2.01mmol). The reaction mixture was stirred at room temperature for 0.5 hourand quickly filtered through celite. The filter was washed withdichloromethane and the organic fraction was concentrated to give thecrude compound T3.

Compound T4:

Without isolation, the crude compound T3 was dissolved in THF (10 mL)followed by the addition of tetrabutylammonium fluoride THF solution(0.0M, 1.0 mL, 1.0 mmol). The mixture was stirring at room temperatureuntil the reaction was complete, which was monitored by TLC. Then, themixture was concentrated in vacuo, saturated NaHCO₃ solution (50 mL) wasadded and the mixture was extracted with dichloromethane. The organiclayer was dried over anhydrous Na₂SO₄, filtered, concentrated and theobtained crude mixture was purified by flash column chromatography(dichloromethane/methanol: 20/1) to give compound T4 (199 mg, 27% fromcompound T2). ¹H NMR (400 MHz, CDCl₃) δ 9.41 (s, 1H), 7.44 (s, 1H), 7.07(t, J=6.6 Hz, 1H), 6.11 (t, J=7.0 Hz, 1H), 4.88-4.80 (m, 2H), 4.57 (m,1H), 4.14 (q, J=2.9 Hz, 1H), 3.93 (m, 1H), 3.82 (m, 1H), 3.49 (d, J=6.2Hz, 2H), 3.10 (t, J=6.2, 4.1 Hz, 1H), 2.42-2.39 (m, 2H), 1.91 (s, 3H),1.31 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 164.39, 158.22, 150.95, 137.33,111.61, 87.33, 85.30, 80.39, 78.65, 77.66, 62.84, 50.70, 48.24, 37.28,25.74, 12.86: MS (APCI⁺) calc'd for C₁₇H₂₄F₃N₃O₆S₂: 487.51, found:487.6.

3′-O—NH₂-DTM-dTTP (T5):

Compound T4 (50 mg, 103 pmol), tetrabutylammonium pyrophosphate (150 mg,0.27 mmol) and 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (33 mg,0.17 mmol) were dried separately over night under high vacuum at ambienttemperature. The tetrabutylammonium pyrophosphate was dissolved indimethylformamide (DMF, 1 mL) under argon followed by addition oftributylamine (1.5 mL). The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of 3′-O-ethyldithiomethyl thymidine and stirred further for 1hour at room temperature. Iodine solution (0.02 M iodine/pyridine/water)was then injected into the reaction mixture until a permanent browncolor was observed. After 10 min, Water (30 mL) was added and thereaction mixture was stirred at room temperature for additional 2 hour.The resulting solution was extracted with ethyl acetate. The aqueouslayer was concentrated in vacuo and the residue was diluted with 5 ml ofwater. The crude mixture was then purified with anion exchangechromatography on DEAE-Sephadex A-25 at 4° C. using a gradient of TEAB(pH 8.0; 0.1-1.0 M). The crude product was further purified byreverse-phase HPLC to afford T5, which was characterized by MALDI-TOFMS, calc'd for C₁₅H₂₈N₃O₁₄P₃S₂: 631.45. found: 631.0.

3′-O-Bodipy-DTM-dTTP (compound T6):

To a stirred solution of Bodipy FL-NHS ester (1.5 mg, 3.9 pmol) in DMF(0.2 ml), 3′-O-DTM-dTTP (compound T5, 4.0 μmol) in NaHCO₃/Na₂CO₃ buffer(pH 8.9, 0.1 M, 0.3 ml). The reaction mixture was stirred at roomtemperature for 3 h with exclusion of light. The reaction mixture waspurified by a preparative silica gel TLC plate(dichloromethane/methanol, 4:1). The crude product was then purifiedwith anion exchange chromatography on DEAE-Sephadex A-25 at 4° C. usinga gradient of TEAB (pH 8.0; 0.1-1.0 M), and further purified onreverse-phase HPLC to afford 3′-O-DTM-Bodipy-dTTP T6, which wascharacterized by MALDI-TOF MS, calc'd for C₂₉H₄BF₂N₅O₁₅P₃S₂: 905.5,found: 904.1.

3′-O-Bodipy-PEG₄-DTM-dTTP (compound T7):

To a stirred solution of Bodipy-PEG₄-Acid (2.1 mg, 3.8 pmol) in dry DMF(200 μl), N,N-disuccinimidyl carbonate (1.03 mg, 4.0 μmol) and4-dimethylaminopyridine (0.48 mg, 4.0 pmol) were added. The reactionmixture was stirred at room temperature for 2 h. TLC indicated thatBodipy-PEG₄-Acid was completely converted to compound Bodipy-PEG₄-NHSester, which was directly used to couple with amino-3′-O-DTM-dTTP (3.8pmol) in NaHCO₃/Na₂CO₃ buffer (pH 8.9, 0.1 M) (300 μl). The reactionmixture was stirred at room temperature for 3 h with exclusion of light.The reaction mixture was purified by a preparative silica gel TLC plate(dichloromethane/methanol, 4:1). The crude mixture was purified withanion exchange chromatography on DEAE-Sephadex A-25 at 4° C. using agradient of TEAB (pH 8.0; 0.1-1.0 M), and further purified onreverse-phase HPLC to afford 3′-O-DTM-PEG₄-Bodipy-dTrP T7, which wascharacterized by MALDI-TOF MS calc'd for C₄₀H₆₂BF₂N₆O₂₀P₃S₂: 1152.8,found: 1151.4.

The Synthesis of 3′-O-Rox-DTM-dATP and 3′-O-Rox-PEG₄-DTM-dATP

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine(A2): To a solution of theN⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine (A1, 1.41 g, 3mmol) in DMSO (10 mL) with stirring was added acetic acid (3 mL) andacetic anhydride (9 mL). The reaction mixture was stirred at roomtemperature until the reaction was complete (48 h), which was monitoredby TLC. Then the mixture was added slowly to the solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the residue of the desired compound was purified by silicagel column chromatography (dichloromethane/methanol: 30/1) to give pureproduct A2 (1.39 g, 88%). ¹H NMR (400 MHz, CDCl₃) δ 9.12 (s, 1H), 8.81(s, 1H), 8.35 (s, 1H), 8.10-8.01 (m, 2H), 7.68 (m, 1H), 7.49 (m, 2H),6.53 (dd, J=7.5, 6.0 Hz, 1H), 4.78-4.65 (m, 3H), 4.24 (dt, J=4.3, 3.1Hz, 1H), 3.98-3.81 (m, 2H), 2.80-2.60 (m, 2H), 2.21 (s, 3H), 0.94 (s,10H), 0.13 (s, 6H); MS (APCI⁺) calc'd for C₂₅H₃₅N₅O₄SSi: 529.73. found:529.4.

Compound A3:

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine(A2, 550 mg, 1.04 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.3 mL) and molecular sieves(3 Å, 2 g). The mixture was cooled in an ice-bath after stirring at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.12mL, 1.50 mmol) in anhydrous dichloromethane (3 mL) was added dropwiseduring 2 minutes. The ice-bath was removed and the reaction mixture wasstirred further for 0.5 hour. Then potassium p-toluenethiosulfonate(0.61 g, 2.25 mmol) in anhydrous DMF (3 mL) was added to the mixture.Stirring was continued at room temperature for additional 1 hourfollowed by addition of2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide (302 mg, 1.5mmol). The reaction mixture was stirred at room temperature for 0.5 hourand quickly filtered through celite. The filter was washed withdichloromethane and the organic fraction was concentrated to give thecrude compound A3: MS (APCI⁺) calc'd for C₃₀H₄₁F₃N₆O₅S₂Si: 714.89,found: 714.6.

Compound A4:

Without isolation, the crude compound A3 was dissolved in THF (10 mL)followed by the addition of tetrabutylammonium fluoride THF solution(1.0 M, 1.0 mL, 1.0 mmol). The mixture was stirred at room temperatureuntil the reaction was complete, which was monitored by TLC. Then, themixture was concentrated in vacuo, saturated NaHCO₃ solution (50 mL) wasadded and the mixture was extracted with dichloromethane. The organiclayer was dried over anhydrous Na₂SO₄, filtered, concentrated and theobtained crude mixture was purified by flash column chromatography(dichloromethane/methanol: 20/1) to give compound A4 (128 mg, 20% fromcompound A2). ¹H NMR (400 MHz, CDCl₃) δ 9.16 (s, 1H), 8.77 (s, 1H), 8.11(s, 1H), 8.07-8.00 (m, 2H), 7.61 (m, 1H), 7.56-7.52 (m, 2H), 6.91 (m,1H), 6.33 (dd, J=9.4, 5.5 Hz, 1H), 5.83 (d, J=10.7 Hz, 1H), 4.88 (d,J=2.6 Hz, 2H), 4.75 (dt, J=5.4, 1.2 Hz, 1H), 4.36 (q, J=1.7 Hz, 1H),4.03 (dd, J=12.8, 1.8 Hz, 1H), 3.81 (t, J=10.9 Hz, 1H), 3.51 (d, J=6.2Hz, 2H), 3.10 (m, 1H), 2.56-2.46 (m, 1H), 1.36 (s, 6H); ¹³C NMR (75 MHz,CDCl3) δ 164.91, 152.49, 151.03, 150.71, 142.95, 133.82, 133.33, 129.29,128.29, 125.00, 118.23, 114.41, 88.01, 87.10, 80.37, 80.19, 63.91,60.76, 50.66, 47.99, 38.17, 25.82, 25.75; MS (APCI⁺) calc'd forC₂₄H₂₇F₃N₆O₅S₂: 600.6, found: 600.7.

3′-NH₂-DTM-dATP (A5):

Compound A4 (50 mg, 103 mmol), tetrabutylammonium pyrophosphate (150 mg,0.27 mmol) and 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (33 mg,0.17 mmol) were dried separately over night under high vacuum at ambienttemperature. The tetrabutylammonium pyrophosphate was dissolved indimethylformamide (DMF, 1 mL) under argon followed by addition oftributylamine (1.5 mL). The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of 3′-O-ethyldithiomethyl thymidine and stirred further for 1hour at room temperature. Iodine solution (0.02 M iodine/pyridine water)was then injected into the reaction mixture until a permanent browncolor was observed. After 10 min, Water (30 mL) was added and thereaction mixture was stirred at room temperature for additional 2 hour.The resulting solution was extracted with ethyl acetate. The aqueouslayer was concentrated in vacuo to approximately 20 mL, thenconcentrated NH₄OH (20 ml) was added and stirred overnight at roomtemperature. The resulting mixture was concentrated under vacuum and theresidue was diluted with 5 ml of water. The crude mixture was thenpurified with anion exchange chromatography on DEAE-Sephadex A-25 at 4°C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude product wasfurther purified by reverse-phase HPLC to afford A5, which wascharacterized by MALDI-TOF MS, calc'd for C₁₅H₂₇N₆O₁₂P₃S₂: 640.45,found: 639.6.

3′-O-Rox-DTM-dATP (compound A6):

To a stirred solution of ROX-NHS ester (2 mg, 3.2 pmol) in DMF (0.2 ml),amino 3′-O-DTM-dATP (compound A5, 3.0 μmol) in NaHCO₃/Na₂CO₃ buffer (pH8.9, 0.1 M, 0.3 ml). The reaction mixture was stirred at roomtemperature for 3 h with exclusion of light. The reaction mixture waspurified by a preparative silica gel TLC plate(dichloromethane/methanol, 4:1). The crude product was then purifiedwith anion exchange chromatography on DEAE-Sephadex A-25 at 4° C. usinga gradient of TEAB (pH 8.0; 0.1-1.0 M), and further purified onreverse-phase HPLC to afford 3′-O-DTM-Rox-dATP A6, which wascharacterized by MALDI-TOF MS, calc'd for C₄₈H₅₅N₈O₁₆P₃S₂: 1157.0,found: 1155.4.

3′-O-Rox-PEG₄-DTM-dATP (compound A7):

To a stirred solution of ROX-PEG₄-Acid (2.6 mg, 3.3 pmol) in dry DMF(200 μl), N,N-disuccinimidyl carbonate (0.90 mg, 3.5 μmol) and4-dimethylaminopyridine (0.43 mg, 3.5 pmol) were added. The reactionmixture was stirred at room temperature for 2 h. TLC indicated thatROX-PEG₄-Acid was completely converted to compound ROX-PEG₄-NHS ester,which was directly used to couple with amino-3′-O-DTM-dATP (3.5 pmol) inNaHCO₃, Na₂CO₃ buffer (pH 8.9, 0.1 M) (3(00) p). The reaction mixturewas stirred at room temperature for 3 h with exclusion of light. Thereaction mixture was purified by a preparative silica gel TLC plate(dichloromethane/methanol, 4:1). The crude mixture was then purifiedwith anion exchange chromatography on DEAE-Sephadex A-25 at 4° C. usinga gradient of TEAB (pH 8.0; 0.1-1.0 M), and further purified onreverse-phase HPLC to afford 3′-O-DTM-PEG₄-Rox-dATP A7, which wascharacterized by MALDI-TOF MS, calc'd for C₅₉H₇₆N₉O₂₁P₃S₂: 1404.3,found: 1401.6.

The Synthesis of 3′-O-Alexa488-DTM-dCTP and 3′-O-PEG₄-Alexa488-DTM-dCTP

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C2): To a solution of theN⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine (C1, 2.25 g,2.51 mmol) in DMSO (20 mL) with stirring was added acetic acid (8 mL)and acetic anhydride (20 mL). The reaction mixture was stirred at roomtemperature until the reaction was complete (24 h), which was monitoredby TLC. Then the mixture was added slowly to the solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×50 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the residue of the desired compound was purified by silicagel column chromatography (DCM/MeOH: 1/30) to give pure product C2 (2.13g, 83%). ¹H NMR (400 MHz, CDCl₃) δ: 8.43 (d, J=8.4 Hz, 1H), 7.92 (d,J=7.6 Hz, 2H), 7.66 (m, 1H), 7.53 (m, 3H), 6.30 (t, J=6.0 Hz, 1H), 4.69(dd, J=32 Hz; 7.6 Hz, 2H), 4.50 (m, 1H), 4.18 (m, 1H), 3.98-3.83 (m,2H), 2.74 (m, 1H), 2.21-2.12 (m, 4H), 0.93 (s, 9H), 0.15 (m, 6H). MS(APCI⁺⁾ calc'd for C₂₄H₃₅N₃O₅SSi: 505.70, found: 505.6.

Compound C3:

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C2, 0.87 mg, 1.72 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.3 mL) and molecular sieves(3 Å, 2 g). The mixture was cooled in an ice-bath after stirring at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.15mL, 1.80 mmol) in anhydrous dichloromethane (3 mL) was added dropwiseduring 2 minutes. The ice-bath was removed and the reaction mixture wasstirred further for 0.5 hour. Then potassium p-toluenethiosulfonate(0.68 g, 2.5 mmol) in anhydrous DMF (3 mL) was added to the mixture.Stirring was continued at room temperature for additional 1 hourfollowed by addition of 2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide (402 mg, 2.0 mmol). The reaction mixture was stirred at roomtemperature for 0.5 hour and quickly filtered through celite. The filterwas washed with dichloromethane and the organic fraction wasconcentrated to give the crude compound C3: MS (APCI⁻) calc'd forC₂₉H₄₁F₃N₄O₆S₂Si: 690.87, found: 689.8 [M−H].

Compound C4:

Without the isolation, the crude compound C3 was dissolved in THF (10mL) followed by the addition of tetrabutylammonium fluoride THF solution(1.0M, 1.0 mL, 1.0 mmol). The mixture was stirring at room temperatureuntil the reaction was complete, which was monitored by TLC. Then, themixture was concentrated in vacuo, saturated NaHCO₃ solution (50 mL) wasadded and the mixture was extracted with dichloromethane. The organiclayer was dried over anhydrous Na₂SO₄, filtered, concentrated and theobtained crude mixture was purified by flash column chromatography(dichloromethane/methanol: 20/1) to give compound C4 (171 mg, 17% fromcompound C2). ¹H NMR (400 MHz, CDCl₃) δ 8.94 (br, 1H), 8.32 (d, J=7.5Hz, 1H), 7.90-7.83 (m, 2H), 7.65-7.55 (m, 2H), 7.49 (dd, J=8.4, 7.1 Hz,2H), 7.12 (t, J=6.3 Hz, 1H), 6.15 (t, J=6.4 Hz, 1H), 4.93-4.78 (m, 2H),4.58 (dt, J=6.5, 3.3 Hz, 1H), 4.24 (q, J=3.0 Hz, 1H), 4.02 (dd, J=12.1,3.0 Hz, 1H), 3.86 (dd, J=12.1, 2.9 Hz, 1H), 3.66 (br, 1H), 3.50 (d,J=6.2 Hz, 2H), 2.71 (m, 1H), 2.40 (m, 1H), 1.34 (s, 6H). ¹³C NMR (75MHz, CDCl₃) δ 162.82, 158.20, 157.72, 155.45, 146.01, 133.63, 129.40,127.98, 97.24, 89.14, 86.04, 80.46, 78.25, 62.47, 50.72, 48.04, 38.47,25.78, MS (APCI⁺) calc'd for C₂₃H₂₇F₃N₄O₆S₂: 576.61, found: 576.

3′-NH₂-DTM-dCTP (C5):

Compound C4 (50 mg, 87 mmol), tetrabutylammonium pyrophosphate (140 mg,0.25 mmol) and 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (30 mg,0.15 mmol) were dried separately over night under high vacuum at ambienttemperature. The tetrabutylammonium pyrophosphate was dissolved indimethylformamide (DMF, 1 mL) under argon followed by addition oftributylamine (1.5 mL). The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of 3′-O-ethyldithiomethyl thymidine and stirred further for 1hour at room temperature. Iodine solution (0.02 M iodine/pyridine/water)was then injected into the reaction mixture until a permanent browncolor was observed. After 10 min, Water (30 mL) was added and thereaction mixture was stirred at room temperature for additional 2 hour.The resulting solution was extracted with ethyl acetate. The aqueouslayer was concentrated in vacuo and the residue was diluted with 5 ml ofwater. The crude mixture was then purified with anion exchangechromatography on DEAE-Sephadex A-25 at 4° C. using a gradient of TEAB(pH 8.0: 0.1-1.0 M), and further purified by reverse-phase HPLC toafford C5, which was characterized by MALDI-TOF MS, calc'd forC₁₄H₂₉N₄O₁₃P₃S₂: 618.4, found: 616.7.

3′-O-Alexa488-DTM-dCTP and 3′-O-PEG₄-Alexa488-DTM-dCTP can besynthesized by coupling the 3′-NH₂-DTM-dCTP (C5) with the NHS ester ofAlexa488.

The Synthesis of 3′-O-Cy5-DTM-dGTP and 3′-O-Cy5-PEG₄-DTM-dGTP

N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine (G2): The mixtureof 2′-deoxyguanosine (G1, 1.33 g, 5 mmol), tert-butyldimethylsilylchloride (825 mg, 5.5 mmol) and imidazol (370 mg, 5.5 mmol) wasdissolved in dry DMF (20 mL) and stirring at room temperature until thereaction was complete, which was monitored by TLC. Then the solvent wasremoved and the residue was added N,N-dimethylformamide dimethyl acetal(2.5 mL) in dry DMF (10 mL). Stirring was continued at room temperaturefor additional 10 hours, then the reaction mixture was poured intostirred ice-water (200 mL) and the precipitate was collected by suctionfiltration, washed with water and hexane. The obtained crude product waspurified by column chromatography (dichloromethane/methanol: 20/1) togive N⁴-DMF-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine (G2, 1.76 g,81%). ¹H NMR (400 MHz, CDCl₃) δ 9.52 (s, 1H), 8.60-8.55 (m, 1H), 7.91(s, 1H), 6.43 (t, J=6.7 Hz, 1H), 4.68 (d, J=4.6 Hz, 1H), 4.16-4.08 (m,1H), 3.94-3.87 (m, 1H), 3.87-3.77 (m, 2H), 3.16 (s, 3H), 3.07 (s, 3H),2.63-2.49 (m, 2H), 0.91 (s, 9H), 0.09 (d, J=0.8 Hz, 6H). MS (APCI⁺): MS(APCI⁺) calc'd for C₉H₃₂N₆O₄Si: 436.58, found: 436.6.

N⁴-DMF-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine(G3): To a solution of theN⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine (G2, 1.31 g, 3mmol) in DMSO (10 mL) with stirring was added acetic acid (3 mL) andacetic anhydride (9 mL). The reaction mixture was stirred at roomtemperature until the reaction was complete (48 h), which was monitoredby TLC. Then the mixture was added slowly to the solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the residue of the desired compound was purified by silicagel column chromatography (dichloromethane/methanol: 30/1) to give pureproduct G3 (1.16 g, 78%). ¹H NMR (400 MHz, CDCl₃) δ 9.76 (s, 1H), 8.61(s, 1H), 7.85 (s, 1H), 6.33 (dd, J=7.4, 6.4 Hz, 1H), 4.74-4.63 (m, 2H),4.63-4.58 (m, 1H), 4.13 (m, 1H), 3.84-3.71 (m, 2H), 3.19 (d, J=0.7 Hz,3H), 3.10 (d, J=0.7 Hz, 3H), 2.58-2.46 (m, 2H), 2.17 (s, 3H), 0.91 (s,9H), 0.09 (s, 6H); MS (APCI⁺) calc'd for C₂₁H₃₆N₆O₄SSi: 496.7, found:496.8. The target compounds 3′-O-Cy5-DTM-dGTP and 3′-O-Cy5-PEG₄-DTM-dGTPare produced.

Consecutive Polymerase Extension Using 3′-O-Rox-DTM-dATP ReversibleTerminator and Characterization by MALDI-TOF Mass Spectrometry (Resultsare Shown in FIG. 33

The first extension reaction was carried out using 200 pmol ofreversible terminator (3′-O-Rox-DTM-dATP), 2 units of Therminator™ IXDNA Polymerase (A 9°N™ DNA Polymerase variant from NEB), 100 pmol of DNAprimer (5′-TAGATGACCCTGCCTTGTCG-3′) (SEQ ID NO:2), 60 pmol of DNAtemplate (5′-GAAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTTGTGCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCITTTCTCTTCGTTCTCCGT-3′) (SEQ ID NO: 1)in a 20 μl buffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl,2 mM MgSO₄, 0.1% Triton X-100. pH 8.8 @ 25° C., and 2 mM MnCl₂. Thereaction was conducted in an ABI GeneAmp PCR System 9700 with initialincubation at 65° C. for 30 second, followed by 38 cycles of 65° C./30sec, 45° C./30 sec, 65° C./30 sec. Multiple reactions were carried outand an aliquot of the reaction mixture was desalted using a C18 ZipTipcolumn (Millipore, MA) and analyzed by MALDI-TOF MS (ABI Voyager DE).

Calf Intestinal Alkaline Phosphatase (CIP) from NEB was used toinactivate residual reversible terminator nucleotide and THP(Tris-hydroxypropyl-phosphine) was used to remove the Rox-tBu-SS groupfrom the 3′ end of the DNA extension product to regenerate the 3′-OHgroup in preparation for the next extension reaction. The cleavagereaction was carried out by incubating the extension reaction mixturewith THP at 5 mM final concentration and incubating at 65° C. for 5minutes.

The reaction mixture after THP treatment was purified by reverse phaseHPLC on an XTerra MS C18, 2.5 μm 4.6 mm×50 mm column (Waters, MA) toobtain the pure cleavage product. Mobile phase: A, 8.6 mMtriethylamine/100 mM 1,1,1,3,3,3-hexafluoro-2-propanol in water (pH8.1); B, methanol. Elution was performed at 40° C. with a 0.5 mL/minflow rate with a linear gradient from 88% A/12% B to 65.5% A/34.5% B for90 min. The purified product was used in the subsequent extensionreaction.

Since there are two consecutive Ts on the DNA template after the DNAprimer binding site, the second extension reaction was carried out inthe same way as the first extension reaction. The overall results areshown in FIG. 33 MALDI TOF MS of the primers after each step demonstrateaccurate incorporation of 3′-Rox-SS-dATP, efficient cleavage of SS bondand recovering of 3′OH, and incorporation of another 3′-Rox-SS-dATP.

DNA Polymerase Extension Using 3′-O-Rox-PEG₄-DTM-dATP ReversibleTerminator. Cleavage Reaction Using THP, and Characterization byMALDI-TOF Mass Spectrometry (Results are Shown in FIGS. 34A-34C.

The DNA Polymerase extension was carried out using 200 pmol ofreversible terminator (3′-O-Rox-PEG₄-DTM-dATP), 2 units of Terminator™IX DNA Polymerase (NEB), 100 pmol of primer (5′-TAGATGACCCTGCCTTGTCG-3′)(SEQ ID NO:2), 60 pmol of DNA template(5′-GAAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTTTGTGCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTTTTCTCTTC GTCTCCGT-3′)(SEQ ID NO:3) in a 20 μl buffer containing 20 mM Tris-HCl, 10 mM(NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH 8.8 a 25° C.,and 2 mM MnCl₂. The reaction was conducted in an ABI GeneAmp PCR System9700 with initial incubation at 65° C. for 30 second, followed by 38cycles of 65° C./30 sec, 45° C./30 sec, 65° C./30 sec. The reactionmixture was then desalted using a C18 ZipTip column (Millipore, MA) andanalyzed by MALDI-TOF MS (ABI Voyager DE).

Calf Intestinal Alkaline Phosphatase (CIP) from NEB was used toinactivate residual reversible terminator nucleotide and THP was used toremove the blocking group from the 3′ end of the DNA extension productto regenerate the 3′-OH group. The cleavage reaction was carried out byincubating the extension reaction mixture with THP at 5 mM finalconcentration and incubating at 65° C. for 5 minutes.

DNA Polymerase Extension Using Either 3′-O-Bodipy-DTM-dTTP, or3′-O-Bodipy-PEG₄-DTM-dTTP Reversible Terminator. Cleavage Reaction UsingTHP, and Characterization by MALDI-TOF Mass Spectrometry (Results areShown in FIGS. 35A-35C and FIGS. 36A-36C

Binary structures of DNA bound to 9° N and the closely related KOD DNApolymerase from Thermococcus kodakaraensis have been published (BergenK, Betz K, Welte W, Diederichs K, Marx A. Structures of KOD and 9° N DNApolymerases complexed with primer template duplex. Chem Bio Chem. 2013;14(9): 1058-1062.) and these authors have speculated on the reasonsthese enzymes may be more tolerant toward modified nucleotides. Theminor groove appears to be relatively less sterically hindered thanfamily A polymerases, perhaps explaining their relative ease of theformer in utilizing nucleotides with small C4′ modifications. Similarly,there may be a more accessible major groove, which could explain theability of these enzymes to accept nucleotides with bulky modificationsat the C5 position of pyrimidines and the C7 position of 7-deazapurines.Unfortunately, crystal structures of ternary complexes with the archaealfamily B polymerases have yet to be obtained, so little can be said withcertainty regarding the positions around an incoming nucleotide, andcrystals of the mutated versions of the 9° N (e.g., Therminator III andIX) have not been published. We have successfully used several of the 9°N polymerase mutants to incorporate deoxynucleotide analogues with awide variety of sometimes quite substantial modifications on theterminal phosphate (Therminator γ (Kumar S, Tao C, Chien M, et al.PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNASequencing by Synthesis. Scientific Reports. 2012:2:684.doi:10.1038/srep00684., Fuller CW, Kumar S, Porel M, et al. Real-timesingle-molecule electronic DNA sequencing by synthesis usingpolymer-tagged nucleotides on a nanopore array. Proceedings of theNational Academy of Sciences of the United States of America. 2016;113(19):5233-5238. doi:10.1073/pnas.1601782113) and base (9° Npolymerase (exo-) A485L/Y409V (Guo J, Xu N, Li Z, et al. Four-color DNAsequencing with 3′-O-modified nucleotide reversible terminators andchemically cleavable fluorescent dideoxynucleotides. Proceedings of theNational Academy of Sciences of the United States of America.2008:105(27):9145-9150. doi: 10.1073/pnas.0804023105, Ruparel et al20105) and Therminator II) as well as a broad range of modifications atthe 3′ oxygen of the sugar (Therminator III and more recentlyTherminator IX).

The DNA Polymerase extension was carried out using 200 pmol ofreversible terminator (3′-O-Bodipy-DTM-dTTP, or3′-O-Bodipy-PEG4-DTM-dTTP), 2 units of Terminator™ IX DNA Polymerase(NEB), 100 pmol of primer (5′-GATAGGACTCATCACCA-3′), (SEQ ID NO:4) 60pmol of DNA template(5′-GAAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTTTGTGCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCITITCTCTTCGTTCTCCGT-3′) (SEQ ID NO:5)in a 20 μl buffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl,2 mM MgSO₄, 0.1% Triton X-100, pH 8.8 @ 25° C., and 2 mM MnCl₂. Thereaction was conducted in an ABI GeneAmp PCR System 9700 with initialincubation at 65° C. for 30 second, followed by 38 cycles of 65° C./30sec, 45° C./30 sec, 65° C./30 sec. The reaction mixture was desaltedusing a C18 ZipTip column (Millipore, MA) and analyzed by MALDI-TOF MS(ABI Voyager DE).

Calf Intestinal Alkaline Phosphatase (CIP) from NEB was used toinactivate residual reversible terminator nucleotide in the extensionreaction mixture and THP was used to remove the blocking group from the3′ end of the DNA extension product to regenerate the 3′-OH group. Thecleavage reaction was carried out by incubating the extension reactionmixture with THP at 5 mM final concentration and incubating at 65° C.for 5 minutes. The reaction mixture was desalted using a C18 ZipTipcolumn (Millipore, MA) and analyzed by MALDI-TOF MS (ABI Voyager DE).

REFERENCES FOR EXAMPLE 1

1. Hyman E. D. (1988) A new method of sequencing DNA. Anal Biochem174(2): 423-436., 2. Ronaghi M. Uhlén M, Nyrdn P (1998) A sequencingmethod based on real-time pyrophosphate. Science 281(5375): 363-365., 3.Ju J. Li Z. Edwards J. R., Itagaki Y (2003) U.S. Pat. No. 6,664,079., 4.Li Z, et al. (2003) A photocleavable fluorescent nucleotide for DNAsequencing and analysis. Proc. Natl. Acad. Sci. USA, 100(2): 414-419.,5. Braslavsky I. Hebert B, Kartalov E, Quake S (2003) Sequenceinformation can be obtained from single DNA molecules. Proc. Natl. Acad.Sci. USA 100(7): 3960-3964., 6. Ruparel H, et al. (2005) Design andsynthesis of a 3′-O-allyl photocleavable fluorescent nucleotide as areversible terminator for DNA sequencing by synthesis. Proc. Natl. Acad.Sci. USA 102(17): 5932-5937., 7. Margulies M. et al. (2005) Genomesequencing in microfabricated high-density picolitre reactors. Nature437(7057): 376-380., 8. Ju J, et al. (2006) Four-color DNA sequencing bysynthesis using cleavable fluorescent nucleotide reversible terminators.Proc. Natl. Acad. Sci. USA 103(52): 19635-19640., 9. Wu J. et al. (2007)3′-O-modified nucleotides as reversible terminators for pyrosequencing.Proc. Natl. Acad. Sci. USA 104(104): 16462-16467., 10. Guo J, et al.(2008) Four-color DNA sequencing with 3′-O-modified nucleotidereversible terminators and chemically cleavable fluorescentdideoxynucleotides. Proc. Natl. Acad. Sci. USA 105(27): 9145-9150., 11.Bentley D. R., et al. (2008) Accurate whole human genome sequencingusing reversible terminator chemistry. Nature 456(7218): 53-59., 12.Harris T. D., et al. (2008) Single-molecule DNA sequencing of a viralgenome. Science 320(5872): 106-109., 13. Eid J, et al. (2009) Real-timeDNA sequencing from single polymerase molecules. Science 323(5910):133-138., 14. Rothberg J. M., et al. (2011) An integrated semiconductordevice enabling non-optical genome sequencing. Nature 475(7356):348-352., 15. Palla M, et al. (2014). DNA sequencing by synthesis using3′-O-azidomethyl nucleotide reversible terminators and surface-enhancedRaman spectroscopic detection. RSC Adv. Jan 1; 4(90): 49342-49346., 16.Hutter D. et al. (2010) Labeled nucleoside triphosphates with reversiblyterminating aminoalkoxy groups. Nucleosides Nucleotides & Nucleic Acids29: 879-895., 17. Knapp D. C., et al. (2011) Fluoride-Cleavable,Fluorescently Labeled Reversible Terminators: Synthesis and Use inPrimer Extension. Chem. Eur. J., 17, 2903-2915., 18. Kwiatkowski M.(2007) Compounds for protecting hydroxyls and methods for their use.U.S. Pat. No. 7,279,563., 19. Muller S, Matthaus J. (2011) Method forproducing trinucleotides. Patent Application WO 2011061114., 20.Semenyuk A, et al. (2006) Synthesis of RNA using 2′-O-DTM protection.JACS, 128, 12356-12357; Ju J, et al (2016) Raman Cluster TaggedMolecules for Biological Imaging. US Patent 20160024570; Ju J, et al(2015) DNA Sequencing by Synthesis Using Raman and Infrared SpectroscopyDetection. US Patent Application 20150080232.

Example 2: Sequencing by Synthesis Methods Using 3′-O-modifiedNucleotide Analogues

Fluorescence-based DNA sequencing-by-synthesis methods have manyadvantages in terms of detection sensitivity. However, because of thelarge size of the fluorophores, specific polymerase and reactionconditions need to be optimized for sequencing reactions. In addition,the current cleavable fluorescent nucleotide reversible terminators usedin SBS leave a modified group, or scar, on the base of the growing DNAstrand after cleavage of the fluorophore, which in turn limits readlength.

Fluorescent NRTs with the following blocking groups at the 3′-OH havebeen reported: 3′-O-allyl-dNTP(Bentley (2008)), 3′-O-azidomethyl-dNTPs(Wu (2007); Guo (2008); Bentley (2008)), 3′-O-NH₂-dNTPs (Hunter (2010)),and 3′-O-cyanoethyl-dNTPs (Knapp (2011)), which can be cleaved by Pd(0),tris(2-carboxyethyl)phosphine (TCEP), dilute nitrous acid and fluoride,respectively, to generate the free 3′-OH group.

Various modifications based on 3′-O-alkyldithiomethyl (3′-O-DTM) for thenucleosides (Kwiatkowski (2007); Muller (2011); Semenyuk (2010)) havebeen reported for the synthesis of oligonucleotides. The stability andreductive cleavage leading to hydroxyl production from the O-DTM grouphas also been established (Kwiatkowski (2007): Muller (2011); Semenyuk(2010)), but their utility in DNA sequencing applications has not beenreported. This is because nucleotide analogs with a large fluorescentdye blocking the 3′-OH group were reported to not be incorporated by DNApolymerase in template-directed DNA synthesis.

By the unique chemical design of the cleavable t-butyldithiomethylmoiety attached to a fluorescent dye to block the 3′-OH group of thenucleotide, coupled with specific polymerase reaction conditions, it isherein disclosed that the modified 3′-O-dithiomethyl (3′-O-DTM) is asuccessful reversible linkage group for attaching a fluorescent dyereporter to block the 3′-OH group of the nucleotide for DNA SBS. To thisend, herein disclosed are novel 3′ reversibly labeled nucleotides astraceless reversible terminators, which were designed and synthesizedfor DNA SBS. In these novel nucleotide analogs, only the 3′-OH group ofthe nucleotide is reversibly blocked with a DTM linker, which isattached to the fluorescent label, thus realizing the dual function ofthe 3′-O-modification of the nucleotide, serving as both the reversibleterminator function and the cleavable fluorescence reporter (FIG. 1 andFIGS. 2A-2E).

It is further disclosed herein, that in SBS cycles, such3′-O-Dye-DTM-dNTPs are well recognized by the DNA polymerase,Therminator (9° N DNA polymerase variant), as substrates andincorporated into the growing DNA strand. After determining the identityof the incorporated nucleotide by its fluorescent signal, TCEP orTris(3-hydroxypropyl)phosphine (THP) treatment cleaves the disulfidebond in the DMT moiety leading to both the removal of the fluorescencereporter and the regeneration of the 3′-OH group (FIGS. 3A-3B) to allowfor continuous sequencing. After each incorporation and cleavage, anextended natural DNA strand is produced to allow for the seamlessincorporation of incoming complementary 3′-O-Dye-DTM-dNTPs during SBS.

There are surprising advantages to using 3′-O-Dye-DTM-dNTPs for SBS. Asdisclosed herein, consecutive polymerase extension reaction using3′-O-Dye-DTM-dNTPs with a synthetic template and primer have beencarried out. After single base extension and cleavage of the DTM moietyand the removal of dye from the 3′-O of the DNA extension product, theresulting primer extension product can be further extended with anadditional 3′-O-Dye-DTM-dNTP, leading to a high-yield incorporation withaccurate sequence determination. Because these 3′-O-Dye-DTM-dNTPs do notrequire the attachment of fluorescent labels on the base, theirsynthesis is simpler and therefore more cost effective. In addition, theextended DNA strand is identical to natural DNA. The use of3′-O-Dye-DTM-dNTPs will lead to very long, accurate read lengths forSBS.

Disclosed herein, and explained in greater detail below, are a varietyof new DNA sequencing methods based on the combinatorial use of3′-O-CleavableLinker-Label-dNTPs, 3′-O-CleavableLinker-Anchor-dNTPs and3′-O-CleavableGroup-dNTPs and their orthogonal reporter dye labeledbinding molecule counterparts or cleavable reporter. Use of3′-O-Dye-SS(DTM)-dNTPs, 3′-O-anchor-SS(DTM)-dNTPs and 3′-O-SS(DTM)-dNTPsalong with orthogonal binding molecules conjugated with fluorescent dyes(or conjugated with fluorescent dyes using different cleavable linkages)allows the construction of a wide spectrum of new methods forfour-color, two-color and one-color DNA SBS at the single molecule levelor the ensemble level.

Example A: One-Color DNA SBS (FIGS. 3A-3B)

Scarless one-color SBS using 3′-O-Biotin-SS(DTM)-dNTPs and Cy5 labeledstreptavidin (FIGS. 2A-2E). DNA polymerase incorporation reaction isconducted by using one of the four 3′-O-Biotin-SS-dNTPs, followed by theaddition of the Cy5 labeled streptavidin and imaging to determine DNAsequences as described in STEP 1 through STEP 4 (as shown in FIGS.3A-3B). Each step consists of three parts: (PART a) Polymerase and oneof the four 3′-O-Biotin-SS-dNTPs are added followed by washing: if theadded nucleotide is complementary to the nucleotide on the templateimmediately next to the 3′ end of the primer, then the added nucleotidewill incorporate into the primer to produce a DNA extension product thathas a Biotin at the 3′ end. (PART b) Cy5 labeled streptavidin is added,which will bond to the Biotin at the 3′ end of the DNA extensionproduct. (PART c) After washing away the unbound Cy5 labeledstreptavidin, imaging is performed to detect the Cy5 signal for theidentification of the incorporated nucleotide. Following STEP 4,addition of THP to the DNA extension products will cleave the disulfidebond and regenerate a free 3′-OH group on the 3′ end of the DNAextension products. The process is sequentially repeated, consisting ofSTEP 1 through STEP 4, followed by THP cleavage, for continuing sequencedetermination.

Example B. Four-Color DNA SBS (FIG. 7)

Scarless SBS using 3′-O-“anchor”-SS(DTM)-dNTPs(3′-O-TCO-t-Butyldithiomethyl(SS)-dATP,3′-O-PBA-t-Butyldithiomethyl(SS)-dCTP,3′-O-Biotin-t-Butyldithiomethyl(SS)-dGTP,3′-O-Azido-t-Butyldithiomethyl(SS)-dTTP) (FIG. 4) and fourcorrespondingly matched dye labeled binding molecules (Rox-LabeledTetrazine, Alexa488-Labeled SHA, Cy5-Labeled Streptavidin, andR6G-Labeled Dibenzocyclooctyne) (FIG. 5). DNA polymerase and the four3′-O-“anchor”-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP, 3′-O-PBA-SS-dCTP,3′-O-Biotin-SS-dGTP and 3′-O—N₃-SS-dTTP) are added to the immobilizedprimed DNA template, which enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. After washing away the unincorporated nucleotideanalogues, the dye labeled binding molecules are added, which willspecifically connect with each of the four unique “anchor” moieties atthe 3′-end of each DNA extension product to enable the labeling of eachDNA product terminated with each of the four nucleotide analogues (A, C,G, T) with the four distinct fluorescent dyes. Detection of the uniquefluorescence signal from each of the fluorescent dyes on the DNAproducts allows for the identification of the incorporated nucleotide.Next, treatment of the DNA products with THP cleaves the SS linker,leading to the removal of the fluorescent dye and the regeneration of afree 3′-OH group on the DNA extension product, which is ready for nextcycle of DNA sequencing reaction (as shown in the subsequent steps ofFIG. 7).

FIGS. 6A-6D shows that the formation of the conjugates or complexesbetween DNA products produced by incorporating 3′-O “anchor” labelednucleotides (3′-O-TCO-t-Butyldithiomethyl-dATP,3′-O-PBA-t-Butyldithiomethyl-dCTP, 3′-O-Biotin-t-Butyldithiomethyl-dGTP,3′-O-Azido-t-Butyldithiomethyl-dTTP) with four correspondingly-matchedlabeled binding molecules (Rox-Labeled Tetrazine, Alexa488-Labeled SHA,Cy5-Labeled Streptavidin, and R6G-Labeled Dibenzocyclooctyne).

Example C. Two-Color DNA SBS (FIGS. 13A-13B)

Use of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP &3′-O-BodipyFL-SS-dCTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O—N₃-SS-dTTP &3′-O-TCO-SS-dGTP) and their corresponding dye labeled binding molecules(Rox-Tetrazine & BodipyFL-Dibenzocyclooctyne) to perform 2-color DNA SBS(FIG. 12). DNA polymerase and the four nucleotide analogues(3′-O-Rox-SS-dATP, 3′-O-BodipyFL-SS-dCTP, 3′-O—N₃-SS-dTTP and3′-O-TCO-SS-dGTP) are added to the immobilized primed DNA template,which enables the incorporation of the complementary nucleotide analogueto the growing DNA strand to terminate DNA synthesis (STEP 1). Afterwashing away the unincorporated nucleotide analogues, the fluorescentsignal from Rox and BodipyFL is detected to identify the incorporatednucleotide as A (labeled with Rox) and C (labeled with BodipyFL). Next,the dye labeled binding molecules (Rox-Tetrazine &BodipyFL-Dibenzocyclooctyne) are added to the DNA extension products(STEP 2), which will specifically connect with the two unique “anchor”moieties (TCO and N₃) at the 3′-end of each DNA extension product, toenable the labeling of each DNA product terminated with each of the twonucleotide analogues (G and T) with two distinct fluorescent dyes(labeled with Rox for G and labeled with BodipyFL for T). Detection ofthe unique, newly produced florescence signal from Rox and BodipyFL onthe DNA extension products (in addition to the signal from STEP 1),allows for the identification of the newly-incorporated nucleotides as Gand T respectively. Next, treatment of the DNA products with THP cleavesthe SS linker, leading to the removal of the fluorescent dye and theregeneration of a free 3′-OH group on the DNA extension product (STEP3), which is ready for the next cycle of DNA sequencing reaction (asshown in the subsequent steps of FIGS. 13A-13B).

Use of 3′-O-CleavableLinker-Label-dNTPs,3′-O-CleavableLinker-Anchor-dNTPs and 3′-O-CleavableGroup-dNTPs combinedwith labeled binding molecules that are conjugated with fluorescencedyes via different cleavable linkage allows the construction ofone-color SBS at the single molecule or the ensemble molecule levels.After incorporating the 3′-anchor-DTM-dNTPs and the 3′-DTM-dNTP,treatment with orthogonal labeled binding molecules conjugated withfluorescence dyes (ATTO647N, Cy5, Rox, etc.) via different cleavablelinkages (Azo, Dde, Nitrobenzyl, Dimethylketal, etc.) (FIG. 14) resultsin the labeling of all incorporated 3′-anchor nucleotides at the 3′-endof the DNA extension products due to the specific anchor-bindingmolecule reaction. Sequential and specific cleavage, followed byimaging, are carried out to remove the dye from the 3′-end of the DNAextension products, allowing signal changes to be accurately detected.Each cleavage method only cleaves one type of the linkage which isuniquely attached to one of the labeled binding molecules, thereforeeach cleavage method can be used to encode one of the DNA bases on theircorresponding 3′-O-anchor moiety for that particular nucleotideanalogue. In general, only three of the four DNA bases (A, C, G, T) arerequired to have a label for selective detection. Once the first threeof these bases are labeled, the fourth one does not require a label tobe differentiated from the other three for sequence determination, asexemplified in the following schemes.

Example D. One-Color DNA SBS (FIGS. 16A-16C)

1: In presence of DNA polymerase, three 3′-anchor nucleotides[3′-SS(DTM)N₃-dATP, 3′-SS(DTM)TCO-dTTP, 3′-SS(DTM)Biotin-dCTP] and3′-tButyl-SS(DTM)-dGTP, as shown in FIG. 15 are added to the primed DNAtemplates to allow incorporation into the primer.

2: The fluorescent label (ATTO647N, for example) is attached by addingDBCO-Azo-(—N═N-Linker)-ATTO647N, Tetrazine-Dde(Linker)-ATTO647N,Streptavidin-ATTO647N (as shown in FIG. 15) to the DNA extensionproducts that contain the incorporated 3′-anchor nucleotide analogues,which leads to the labeling of all the incorporated nucleotides (exceptG) at their 3′-end due to specific anchor-binding molecule interaction.

3: After washing, the first round of imaging is performed, and the DNAproducts terminated with A C and T all display the same color, while theDNA products that do not emit a signal are terminated by a nucleotide G.

4: The first cleavage (I) is conducted by treatment with sodiumdithionite (Na₂S₂O₄), which only cleaves the azo linkage to remove thefluorescent dye from the DNA products terminated with the A nucleotide.The second round of imaging is performed. If the fluorescent signaldisappears after the cleavage I, the DNA products are determined ashaving incorporated an A nucleotide.

5: The second cleavage (11) is conducted by treatment with hydrazine(N₂H₄), which will cleave the Dde linkage to remove the fluorescent dyefrom the DNA products terminated with the T nucleotide. The third roundof imaging is performed. If the fluorescent signal disappears after thecleavage II, the DNA products are determined as having incorporated a Tnucleotide. The DNA products with unchanged fluorescent signals areidentified by inference as being terminated by a C nucleotide.

6: The third cleavage (III) is conducted with THP to cleave thedisulfide bond and remove the dye on C, so the change of the signalafter the THP treatment also determines the DNA products as beingterminated by a C nucleotide. Meanwhile, the THP treatment will alsocleave the DTM (SS) bond to regenerate free 3′-OH on all the DNAextension products, which are ready for subsequent cycles ofsingle-color DNA SBS.

7: Repeat steps 1 to 6 to continue subsequent cycles of single-color DNASBS.

Example E. One-Color DNA SBS (FIGS. 18A-18C)

1: In presence of DNA polymerase, two 3′-anchor nucleotides[(3′-O—N₃-SS(DTM)-dTTP, 3′-O-Biotin-SS(DTM)-dCTP)],3′-O-Rox-SS(DTM)-dATP and 3′-O-tButyl-SS(DTM)-dGTP, as shown in FIG. 17]are added to the primed DNA templates to allow incorporation into theprimer.

2: Attach the fluorescent label (Rox, for example) by addingDBCO-Azo-(—N═N-Linker)-Rox, Streptavidin-Rox (as shown in FIG. 17) tothe DNA extension products that contain the incorporated 3′-anchornucleotide analogues, which leads to the labeling of all theincorporated nucleotides (except G) at their 3′-end due to specificanchor-binding molecule interaction.

3: After washing, the second round of imaging is performed, and the DNAproducts terminated with A, C and T all display the same Rox signal,while the DNA products that do not emit a signal is terminated by anucleotide G.

4: The first cleavage (1) is conducted by treatment with sodiumdithionite (Na₂S₂O₄), which only cleaves the azo linkage to remove thefluorescent dye Rox from the DNA products terminated with the Tnucleotide. The second round of imaging is performed. If the Roxfluorescent signal disappears after the cleavage I, the DNA products aredetermined as having incorporated a T nucleotide.

5: The second cleavage (II) is conducted with THP to cleave thedisulfide bond and remove the dye from the DNA extension productsterminated with nucleotides A and C, so the change of the signal afterthe THP treatment determines the DNA products as being terminated by a Cnucleotide, because DNA products as being terminated by an A nucleotidehave already being determined in the first round of imaging describedabove. Meanwhile, the THP treatment will also claeve the DTM (SS) bondto regenerate free 3′-OH on all the DNA extension products, which areready for subsequent cycles of single-color DNA SBS. Repeat steps tocontinue subsequent cycles of single-color DNA SBS.

Example F. One-Color DNA SBS (FIGS. 20A-20C)

1: In presence of DNA polymerase, three 3′-anchor nucleotides[3′-O—N₃-SS(DTM)-dGTP, 3′-O-Biotin-SS(DTM)-dCTP, 3′-O-TCO-SS(DTM)-dTTP)]and 3′-O-Rox-SS(DTM)-dATP, as shown in FIGS. 19A-19B] are added to theprimed DNA templates to allow incorporation into the primer.

2: After washing, the first round of imaging is performed, and the DNAproducts terminated with an A nucleotide analogue display the Rox signaland therefore are determined as having incorporated an A nucleotide,while the other DNA products terminated at G, C, T will not display anyfluorescent signals.

3: Attach the fluorescent label (Rox, for example) by addingDBCO-Azo-(—N═N-Linker)-Rox, Tetrazine-Dde-Rox and Streptavidin-Rox (asshown in FIGS. 19-19B) to the DNA extension products that contain theincorporated 3′-anchor nucleotide analogues, which leads to the labelingof all the incorporated nucleotides at their 3′-end due to specificanchor-binding molecule interaction.

4: After washing, the second round of imaging is performed, and the DNAproducts terminated with A, G, T, C all display the same Rox signal.Subtraction of the Rox signals from the DNA products determined in thefirst round of imaging as terminated at an A nucleotide reveals the DNAproducts terminated at G, T, C.

5: The first cleavage (I) is conducted by treatment with sodiumdithionite (Na₂S₂O₄), which only cleaves the azo linkage to remove thefluorescent dye Rox from the DNA products terminated with the Gnucleotide. The second round of imaging is performed. If the Roxfluorescent signal disappears after the cleavage I, the DNA products aredetermined as having incorporated a G nucleotide.

6: The second cleavage (II) is conducted with hydrazine (N₂H₄), whichwill cleave the Dde linkage to remove the fluorescent dye Rox from theDNA products terminated with the T nucleotide. The third round ofimaging is performed. If the Rox fluorescent signal disappears after thecleavage II, the DNA products are determined as having incorporated a Tnucleotide. If the Rox fluorescent signal stays after the cleavage II,the DNA products are determined as having incorporated a C nucleotide.

7: The third cleavage (III) is conducted with THP to cleave thedisulfide bond and remove the Rox dye from the DNA extension productsterminated with nucleotides A and C, so the change of the signal afterthe THP treatment also determines the DNA products as being terminatedby a C nucleotide, because DNA products as being terminated by an Anucleotide have already being determined in the first round of imagingdescribed above. Meanwhile, the THP treatment will also cleave the DTM(SS) bond to regenerate free 3′-OH on all the DNA extension products,which are ready for subsequent cycles of single-color DNA SBS. Repeatsteps 1 to 7 to continue subsequent cycles of single-color DNA SBS.

Example G One Color DNA SBS (FIG. 22)

(1) In presence of DNA polymerase, the three3′-O-CleavableLinker-Label-dNTPs [3′-O-Rox-SS(DTM)-dATP,3′-O-Rox-Allyl-dTTP, 3′-O-Rox-Nitrobenzyl-dCTP] and 3′-O-tButyl-SS-dGTP,as shown in FIG. 21] are added to the primed DNA templates to allowincorporation into the primer.

(2) After washing, the first round of imaging is performed, and the DNAproducts terminated with C, T and A all display the same Rox signal,while the DNA products that do not emit a signal is terminated by anucleotide G.

(3) The first cleavage (I) is conducted by photo-irradiation at ˜350 nmto remove the fluorescent dye Rox from the DNA products terminated withthe C nucleotide. The second round of imaging is performed. If the Roxfluorescent signal disappears after the cleavage I, the DNA products aredetermined as having incorporated a C nucleotide.

(4) The second cleavage (II) is conducted with Pd (0), which will cleavethe allyl linkage to remove the fluorescent dye Rox from the DNAproducts terminated with the T nucleotide. The third round of imaging isperformed. If the Rox fluorescent signal disappears after the cleavageII, the DNA products are determined as having incorporated a Tnucleotide. If the Rox fluorescent signal stays after the cleavage II,the DNA products are determined as having incorporated an A nucleotide.

(5) The third cleavage (III) is conducted with THP to cleave thedisulfide bond and remove the Rox dye from the DNA extension productsterminated with nucleotides A, so the change of the signal after the THPtreatment also determines the DNA products as being terminated by an Anucleotide. Meanwhile, the THP treatment will also cleave the DTM (SS)bond to regenerate free 3′-OH on all the DNA extension products, whichare ready for subsequent cycles of single-color DNA SBS. Repeat steps 1to 5 to continue subsequent cycles of single-color DNA SBS.

All the above example sequencing methods (Examples A-G) can be modifiedwith unlabeled nucleotide reversible terminator chasing extension (Ju(2006)) using 3′-O-t-Butyl-SS-dNTPs. In this procedure,3′-O-t-Butyl-SS-dNTPs will be used to run polymerase extension aftereach steps of polymerase extension reaction using3′-O-CleavableLinker-Label-dNTPs and 3′-O-CleavableLinker-Anchor-dNTPsto ensure the complete primer extension at the 3′-end for ensemble SBS.

MALDI-TOF mass spectra of the DNA extension products from polymerasereactions using some of the nucleotide analogues described above areperformed and the results are described in FIGS. 23A-23B to FIGS.28A-28C. The results indicate that 3′-O-tButyl-SS-dATP modified with arelatively smaller 3′-O blocking group is incorporated by polymerasewith a much higher efficiency than 3′-O-Rox-SS-dATP labeled with a bulkyRox dye. Nucleotide analogue modified by a Rox through a PEG4 linker isshown to be a better substrate for the DNA polymerase than thenucleotide analogue modified by Rox without a PEG linker.

Polymerase Extension Using 3′-O-DTM-dNTPs, 3′-O-Anchor-DTM-dNTPs and3′-O-Dye-DTM-dNTPs and Characterization by MALDI-TOF Mass Spectrometry

Polymerase extension reaction using 3′-O-SS-Rox-dATP for 5, 10, and 30cycles. The extension reaction was carried out using 200 pmol ofreversible terminator 3′-O-SS-Rox-dATP, 2 units of Therminator IX DNAPolymerase (NEB), 20 pmol of DNA primer (M.W. 6084), 100 pmol of DNAtemplate in a 20 μl buffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄,10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH 8.8 (a 25° C., and 2 mMMnCl₂. The reactions were conducted in an ABI GeneAmp PCR System 9700with initial incubation at 65° C. for 30 second, followed by 5, 10, or30 cycles of 65° C./30 sec, 45° C./30 sec, 65° C./30 sec. The reactionmixtures were desalted using Oligo Clean & Concentrator™ (ZYMO Research)and analyzed by MALDI-TOF MS (ABI Voyager DE) and the results are shownin FIGS. 23A-23B. DNA template:5′-GAAGGAGACACGCGGCCAGAGAGGTCCTGTCCGTGTTTGTCCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTITTCTCTTCGTTCTCCGT-3′ (SEQ ID NO:6).DNA Primer (M.W. 6084): 5′-TAGATGACCCTGCCTTGTCG-3′ (SEQ ID NO:7)

Polymerase extension reaction using 3′-O-tButyl-SS-dATP (5 cycles). Theextension reaction was carried out using 200 pmol of reversibleterminator 3′-O-tButyl-SS-dATP, 2 units of Therminator IX DNA Polymerase(NEB), 20 pmol of DNA primer (M.W. 6084), 100 pmol of DNA template in a20 μl buffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mMMgSO₄, 0.1% Triton X-100, pH 8.8 @ 25° C., and 2 mM MnCl₂. The reactionwas conducted in an ABI GeneAmp PCR System 9700 with initial incubationat 65° C. for 30 second, followed by 5 cycles of 65° C./30 sec, 45°C./30 sec, 65° C./30 sec. The reaction mixtures were desalted usingOligo Clean & Concentrator™ (ZYMO Research) and analyzed by MALDI-TOF MS(ABI Voyager DE) the result is shown in FIG. 24. DNA template:5′-GAAGGAGACACGCGGCCAGAGAGGGTCCGTCCTGTCCGTGGAGTICGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTITTCTCTTCGTTCTCGT-3′ (SEQ ID NO:8).DNA Primer (M.W. 6084): 5′-TAGATGACCCTGCCTTGTCG-3′ (SEQ ID NO:9)

Polymerase extension reaction using 3′-O-tButyl-SS-dATP and3′-O-Rox-SS-dATP at a ratio of 1:1. The extension reaction was carriedout using 100 pmol of reversible terminator 3′-O-tButyl-SS-dATP, 100pmol of reversible terminator 3′-O-Rox-SS-dATP, 2 unit of Therminator IXDNA Polymerase (NEB), 20 pmol of DNA primer (M.W. 6084), 100 pmol of DNAtemplate in a 20 μl buffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄,10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100. pH 8.8 @ 25° C., and 2 mMMnCl₂. The reaction was conducted in an ABI GeneAmp PCR System 9700 withinitial incubation at 65° C. for 30 second, followed by 38 cycles of 65°C./30 sec, 45° C./30 sec, 65° C./30 sec. The reaction mixtures weredesalted using Oligo Clean & Concentrator™ (ZYMO Research) and analyzedby MALDI-TOF MS (ABI Voyager DE) the result is shown in FIGS. 25A-25C.DNA template: 5′-GAAGGAGACACGCGGCCAGAGAGGGTCCTCCTGTCCGTGGAGTICGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTTTTCTCTTCGTTCTCCGT-3′ (SEQ IDNO:10). DNA Primer (M.W. 6084): 5′-TAGATGACCCTGCCTTGTCG-3′ (SEQ ID NO:11).

Polymerase extension reaction using 3′-O-SS-TCO-dTTP. The extensionreaction was carried out using 100 pmol of reversible terminator3′-O-SS-TCO-dTTP, 2 units of Therminator IX DNA Polymerase (NEB), 20pmol of DNA primer (M.W. 5163), 100 pmol of DNA template in a 20 μlbuffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mMMgSO₄, 0.1% Triton X-100, pH 8.8 @ 25° C., and 2 mM MnCl₂. The reactionwas conducted in an ABI GeneAmp PCR System 9700 with initial incubationat 65° C. for 30 second, followed by 38 cycles of 65° C./30 sec, 45°C./30 sec, 65° C./30 sec. The reaction mixtures were desalted usingOligo Clean & Concentrator™ (ZYMO Research) and analyzed by MALDI-TOF MS(ABI Voyager DE) the result is shown in FIG. 26. DNA template:5′-GAAGGAGACACGCGGCCAGAGAGGGTCCGTCCTGTCCGTGTTGTCCGTGGAGTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTITrCTCTTCGTTCTCCGT-3′ (SEQ IDNO:12). DNA Primer (M.W. 5163): 5′-GATAGGACTCATCACCA-3′ (SEQ ID NO: 13).

Polymerase extension reaction using 3′-O-Biotin-SS-dCTP. The extensionreaction was carried out using 10) pmol of reversible terminator3′-O-Biotin-dCTP, 2 units of Therminator IX DNA Polymerase (NEB), 20pmol of DNA primer (M.W. 6131), 100 pmol of DNA template in a 20 μlbuffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mMMgSO₄, 0.1% Triton X-100), pH 8.8 @ 25° C., and 2 mM MnCl₂. The reactionwas conducted in an ABI GeneAmp PCR System 9700 with initial incubationat 65° C. for 30 second, followed by 38 cycles of 65° C./30 sec, 45°C./30 sec, 65° C./30 sec. The reaction mixtures were desalted usingOligo Clean & Concentrator™ (ZYMO Research) and analyzed by MALDI-TOF MS(ABI Voyager DE) the result is shown in FIGS. 27A-27B. DNA template:5′-TACCCGGAGGCCAAGTACGGCGGGTACGTCCTTGACAATGTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGGG-3′ (SEQ ID NO:14). DNAprimer (M.W. 6131): 5′-GTTGATGTACACATTGTCAA-3′ (SEQ ID NO: 15).

Polymerase extension reaction using a mixture of 3′-O-Rox-SS-dATP and3′-O-Rox-PEG₄-SS-dATP at a ratio of 1:1 (5 cycles). The extensionreaction was carried out using 100 pmol of reversible terminator3′-O-Rox-SS-dATP, 100 pmol of reversible terminator(3′-O-Rox-PEG₄-SS-dATP), 2 units of Therminator IX DNA Polymerase (NEB),20 pmol of DNA primer (M.W. 6084), 100 pmol of DNA template in a 20 dbuffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mMMgSO₄, 0.1% Triton X-100, pH 8.8 (25° C. and 2 mM MnCl₂. The reactionwas conducted in an ABI GeneAmp PCR System 9700 with initial incubationat 65° C. for 30 second, followed by 5 cycles of 65° C./30 sec, 45°C./30 sec, 65° C./30 sec. The reaction mixtures were desalted usingOligo Clean & Concentrator™ (ZYMO Research) and analyzed by MALDI-TOF MS(ABI Voyager DE) the result is shown in FIGS. 28A-28C. DNA template:5′-GAAGGAGACACGCGGCCAGAGAGGGTCCTCCTGTCCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTTTTCTCTTCGTTCTCCGT-3′ (SEQ IDNO:16). DNA Primer (M.W. 6084): 5′-TAGATGACCCTGCCTTGTCG-3′ (SEQ ID NO:17).

The structures of 3′-O-tBu-SS-dNTPs are shown in FIG. 29 and thesemolecules are synthesized according the schemes described below.

Synthesis of 3′-O-Modified Nucleotide Analogues

Disclosed herein, and in and explained in greater detail below, is thedesign and synthesis of the three groups of nucleotides with thefollowing general structure 3′-O-CleavableLinker-Label-dNTPs,3′-O-CleavableLinker-Anchor-dNTPs and 3′-O-CleavableGroup-dNTPs aredescribed as follows: 3′-O-DTM-Dye-dNTPs, 3′-O-anchor-DTM-dNTPs (FIGS.2A-2E and FIG. 4) and 3′-O-DTM-dNTPs in which fluorescent dye or a smallanchor moiety is attached to the 3′-O-position of the nucleotide througha DTM cleavable linker. Each incorporated nucleotide analogue contains a3′-O-DTM group that is cleaved after each cycle of sequencedetermination; the 3′-OH of the incorporated nucleotide is thenregenerated for subsequent cycles of SBS. Using MALDI-TOF MS to analyzethe DNA extension products resulting from the use of the abovementionednucleotides in polymerase reactions, we established that these 3′-Omodified nucleotide analogues are efficient substrates for DNApolymerase to terminate the DNA synthesis. These results alsoestablished that both the fluorophore (or anchor moiety) and the3′-O-DTM group are removable with high efficiency in a single step in anaqueous solution and without any residual scars on the incorporatednucleotide. Thus, the natural nucleotides are restored after eachnucleotide incorporation and cleavage, producing a growing DNA strandthat bears no modifications and will not impede further polymerasereactions.

Synthesis of 3′-O-tert-butyldithiomethyl-dTTP (5a) (Scheme 30)

3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl thymidine (T2): To astirring solution of 5′-O-tert-butyldimethylsilyl thymidine (T1, 1.07 g,3 mmol) in DMSO (10 mL) was added acetic acid (2.6 mL, 45 mmol) andacetic anhydride (8.6 mL, 90 mmol). The reaction mixture was stirred atroom temperature until the reaction was complete, which was monitored byTLC. Then the mixture was added slowly to a saturated solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the compound was purified by silica gel columnchromatography (ethyl acetate/hexane: 1:2) to give pure product T2 (0.97g, 74%). ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (s, 1H), 7.48 (s, 1H), 6.28 (m,1H), 4.62 (m, 2H), 4.46 (m, 1H), 4.10 (m, 1H), 3.78-3.90 (m, 2H), 2.39(m, 1H), 2.14 (s, 3H), 1.97 (m, 1H), 1.92 (s, 3H), 0.93 (s, 9H), 0.13(s, 3H); HRMS (FAB⁺) calc'd for C₁₈H₃₃N₂O₅SSi [(M+H)+]: 417.1879, found:417.1890.

3′-O-tert-butyldithiomethyl-5′-O-tert-butyldimethylsilyl thymidine (T3)3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl thymidine (T2, 420mg, 1 mmol) was dissolved in anhydrous dichloromethane (20 mL), followedby addition of triethylamine (0.18 mL, 1.31 mmol, 1.2 eq.) and molecularsieve (3 Å, 2 g). The mixture was cooled in an ice-bath after stirringat room temperature for 30 min and then a solution of sulfuryl chloride(redistilled, 0.1 mL, 1.31 mmol, 1.2 eq.) in anhydrous dichloromethane(3 mL) was added dropwise over 2 minutes. The ice-bath was removed andthe reaction mixture was stirred further for 30 min. Then potassiump-toluenethiosulfonate (375 mg, 1.65 mmol) in anhydrous DMF (2 mL) wasadded to the mixture. Stirring was continued at room temperature foradditional hour followed by addition of tert-butyl mercaptan (1 mL). Thereaction mixture was stirred at room temperature for 30 min and quicklyfiltered through celite. The filter was washed with dichloromethane andthe organic fraction was concentrated to give crude product T3.

3′-O-tert-butyldithiomethyl-thymidine (T4): Without isolation, the crudecompound T3 was dissolved in THF (10 mL) and a THF solution oftetrabutylammonium fluoride(1.0M, 1.04 mL, 1.04 mmol) was added. Thereaction mixture was stirred at room temperature for 4 hours. Thereaction mixture was concentrated in vacuo, saturated NaHCO₃ solution(50 mL) was added and the mixture was extracted with dichloromethane(3×20 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered,concentrated and the obtained crude mixture was purified by flash columnchromatography (dichloromethane/methanol: 20/1) to give3′-O-tert-butyldithiomethyl-thymidine T4 (132 mg, 35% from compound T2).¹H NMR (300 MHz, CDCl₃) δ: 7.41 (q, J=1.2 Hz, 1H), 6.15 (dd, J=7.4, 6.5Hz, 1H), 4.89-4.82 (m, 2H), 4.62-4.54 (m, 1H), 4.15 (q, J=3.0 Hz, 1H),3.97-3.86 (m, 2H), 2.42 (ddd, J=7.5, 4.8, 2.5 Hz, 2H), 1.95 (d, J=1.2Hz, 3H), 1.36 (s, 8H).

3′-O-tert-butyldithiomethyl-dTTP (T5):3′-O-tert-butyldithiomethyl-thymidine (T4, 50 mg, 0.13 mmol),tetrabutylammonium pyrophosphate (197 mg, 0.36 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).This mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of 3′-O-tert-butyldithiomethyl-thymidine and stirred furtherfor 1 hour at room temperature. Iodine solution (0.02 Miodine/pyridine/water) was then injected into the reaction mixture untila permanent brown color was observed. After 10 min, water (30 mL) wasadded and the reaction mixture was stirred at room temperature foradditional 2 hours. The resulting solution was extracted with ethylacetate (2×30 mL). The aqueous layer was concentrated under vacuum andthe residue was diluted with 5 ml of water. The crude mixture was thenpurified with anion exchange chromatography on DEAE-Sephadex A-25 at 4°C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude product wasfurther purified by reverse-phase HPLC to afford T5, which wascharacterized by MALDI-TOF MS calc'd for CI₅H₂₇N₂O₁₄P₃S₂: 616.4, found:615.4.

Synthesis of 3′-O-tert-butyldithiomethyl-dGTP (Scheme 31)

N²-isobutyryl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine(G2): To a stirring solution of theN²-isobutyryl-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine (G1, 1.31g, 3 mmol) in DMSO (10 mL) was added acetic acid (2.6 mL, 45 mmol) andacetic anhydride (8.6 mL, 90 mmol). The reaction mixture was stirred atroom temperature until the reaction was complete, which was monitored byTLC. Then the mixture was added slowly to a saturated solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the compound was purified by silica gel columnchromatography (DCM/methanol: 20:1) to give pure product G2 (75%, 1.15g). ¹H NMR (400 MHz, CDCl₃) δ 12.10 (d, J=2.9 Hz, 1H), 9.17 (d, J=3.0Hz, 1H), 8.03 (m, 1H), 6.18 (td, J=6.9, 2.9 Hz, 1H), 4.74-4.60 (m, 3H),4.13 (dq, J=6.8, 3.3 Hz, 1H), 3.84-3.75 (m, 2H), 2.78 (m, 1H), 2.54 (m,2H), 2.16 (s, 3H), 1.33-1.22 (m, 6H), 0.96-0.87 (m, 9H), 0.09 (dd,J=6.7, 3.8 Hz, 6H).

N²-isobutyryl-3′-O-tert-butyldithiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine(G3):N²-isobutyryl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine(G2, 511 mg, 1.0 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.17 mL, 1.2 mmol) andmolecular sieve (3 Å, 2 g). The mixture was cooled in an ice-bath afterstirring at room temperature for 30 min and then a solution of sulfurylchloride (0.095 mL, 1.2 mmol) in anhydrous dichloromethane (3 mL) wasadded dropwise over 2 minutes. The ice-bath was removed and the reactionmixture was stirred further for 30 min. Then potassium4-toluenethiosulfonate (341 mg, 1.5 mmol) in anhydrous DMF (2 mL) wasadded to the mixture. Stirring was continued at room temperature for anadditional hour followed by addition of tert-butyl mercaptan (1 mL). Thereaction mixture was stirred at room temperature for 30 min and quicklyfiltered through celite. The filter was washed with dichloromethane andthe organic fraction was concentrated to give crude product G3.

N²-isobutyryl-3′-O-tert-butyldithiomethyl-2′-deoxyguanosine (G4):Without the isolation, the crude compound G3 was dissolved in THF (10mL) and a THF solution of tetrabutylammonium fluoride(1.0M, 1.04 mL,1.04 mmol) was added. The reaction mixture was stirred at roomtemperature for 4 hours. The reaction mixture was concentrated in vacuo,saturated NaHCO₃ solution (50 mL) was added and the mixture wasextracted with dichloromethane (3×20 mL). The organic layer was driedover anhydrous Na₂SO₄, filtered, concentrated and the obtained crudemixture was purified by flash column chromatography(dichloromethane/methanol: 20/1) to giveN²-isobutyryl-3′-O-tert-butyldithiomethyl-2′-deoxyguanosine G4 (155 mg,33% from compound G2). ¹H NMR (400 MHz, CDCl₃) δ 12.19 (s, 1H), 9.44 (s,1H), 7.97 (s, 1H), 6.17 (dd, J=8.4, 5.9 Hz, 1H), 5.04 (s, 1H), 4.92-4.80(m, 2H), 4.76-4.64 (m, 1H), 4.26 (q, J=2.6 Hz, 1H), 3.98 (dd, J=12.2,2.8 Hz, 1H), 3.80 (d, J=12.3 Hz, 1H), 2.91-2.73 (m, 2H), 2.49 (m, 1H),1.35 (s, 9H), 1.36-1.22 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 179.60,155.80, 148.10, 147.96, 139.11, 122.30, 86.29, 81.22, 78.96, 63.21,48.07, 38.18, 36.64, 30.29, 19.39, 19.34.

3′-O-tert-butyldithiomethyl-dGTP (G5):N²-isobutyryl-3′-O-tert-butyldithiomethyl-2′-deoxyguanosine (G4, 50 mg,0.11 mmol), tetrabutylammonium pyrophosphate (180 mg, 0.33 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).This mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of N²-isobutyryl-3′-O-tert-butyldithiomethyl-2′-deoxyguanosineand stirred further for 1 hour at room temperature. Iodine solution(0.02 M iodine/pyridine/water) was then injected into the reactionmixture until a permanent brown color was observed. After 10 min, water(30 mL) was added and the reaction mixture was stirred at roomtemperature for additional 2 hours. The resulting solution was extractedwith ethyl acetate. The aqueous layer was concentrated in vacuo toapproximately 20 mL, Then concentrated NH₄OH (20 ml) was added andstirred overnight at room temperature. The resulting mixture wasconcentrated under vacuum and the residue was diluted with 5 ml ofwater. The crude mixture was then purified with anion exchangechromatography on DEAE-Sephadex A-25 at 4° C. using a gradient of TEAB(pH 8.0; 0.1-1.0 M). The crude product was further purified byreverse-phase HPLC to afford G5. HRMS (ESI−) calc'd forC₁₅H₂₅N₅O₁₃P₃S₂[(M−H)⁻]: 640.0103, found: 640.0148.

Synthesis of 3′-O-tert-butyldithiomethyl-dATP (Scheme 32)

N⁶-Benzoyl-5′-O-tert-butyldimethylsilyl-3′-O-methylthiolmethyl-2′-deoxyadenosine(A2): To a solution of theN⁶-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine (A1, 1.41 g, 3mmol) in DMSO (10 mL) with stirring was added acetic acid (3 mL) andacetic anhydride (9 mL). The reaction mixture was stirred at roomtemperature until the reaction was complete, which was monitored by TLC.Then the mixture was added slowly to the solution of sodium bicarbonateunder vigorous stirring and extracted with ethyl acetate (3×30 mL). Thecombined organic layers were dried over Na₂SO₄ and filtered. Thefiltrate was concentrated to dryness under reduced pressure and theresidue of the desired compound was purified by silica gel columnchromatography (dichloromethane/methanol: 30/1) to give pure product A2(1.39 g, 88%). ¹H NMR (400 MHz. CDCl₃) δ 9.12 (s, 1H), 8.81 (s, 1H),8.35 (s, 1H), 8.10-8.01 (m, 2H), 7.68 (m, 1H), 7.49 (m, 2H), 6.53 (dd,J=7.5, 6.0 Hz, 1H), 4.78-4.65 (m, 3H), 4.24 (dt, J=4.3, 3.1 Hz, 1H),3.98-3.81 (m, 2H), 2.80-2.60 (m, 2H), 2.21 (s, 3H), 0.94 (s, 10H), 0.13(s, 6H); MS (APCI⁺) calc'd for C₂₆H₃₆N₄O₄SSi: 528.74, found: 529.4[M+H]⁺.

N⁶-Benzoyl-5′-O-tert-butyldimethylsilyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine(A3):N⁶-Benzoyl-5′-O-tert-butyldimethylsilyl-3′-O-methylthiolmethyl-2′-deoxyadenosine(A2, 529 mg, 1.0 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.17 mL, 1.2 mmol) andmolecular sieve (3 Å, 2 g). The mixture was cooled in an ice-bath afterstirring at room temperature for 30 min and then a solution of sulfurylchloride (0.095 mL, 1.2 mmol) in anhydrous dichloromethane (3 mL) wasadded dropwise over 2 minutes. The ice-bath was removed and the reactionmixture was stirred further for 30 min. Then potassium4-toluenethiosulfonate (341 mg, 1.5 mmol) in anhydrous DMF (2 mL) wasadded to the mixture. Stirring was continued at room temperature for anadditional hour followed by addition of tert-butyl mercaptan (1 mL). Thereaction mixture was stirred at room temperature for 30 min and quicklyfiltered through celite. The filter was washed with dichloromethane andthe organic fraction was concentrated to give crude product A3.

N⁶-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine (A4): Withoutthe isolation, the crude compound A3 was dissolved in THF (10 mL) and aTHF solution of tetrabutylammonium fluoride(1.0M, 1.04 mL, 1.04 mmol)was added. The reaction mixture was stirred at room temperature for 4hours. The reaction mixture was concentrated in vacuo, saturated NaHCO₃solution (50 mL) was added and the mixture was extracted withdichloromethane (3×20 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered, concentrated and the obtained crude mixture waspurified by flash column chromatography (dichloromethane/methanol: 20/1)to give N⁶-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine A4 (128mg, 26% from compound A2). ¹H NMR (400 MHz, DMSO-d₆) δ 11.18 (s, 1H),8.77 (s, 1H), 8.71 (s, 1H), 8.10-8.02 (m, 2H), 7.66 (t, J=7.6 Hz, 1H),7.56 (t, J=7.6 Hz 2H), 6.47 (dd, J=8.0, 6.0 Hz, 1H), 5.15 (t, J=5.5 Hz,1H), 5.00 (s, 2H), 4.65 (dt, J=5.4, 2.4 Hz, 1H), 4.12 (td. J=4.7, 2.2Hz, 1H), 3.02-2.88 (m, 1H), 2.84 (q, J=7.3 Hz, 2H), 2.61 (m, 1H), 1.35(s, 9H).

3′-O-tert-butyldithiomethyl-dATP (A5):N⁶-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine (A4, 50 mg,0.10 mmol), tetrabutylammonium pyrophosphate (180 mg, 0.33 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).This mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of N⁶-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine andstirred further for 1 hour at room temperature. Iodine solution (0.02 Miodine, pyridine/water) was then injected into the reaction mixtureuntil a permanent brown color was observed. After 10 min, water (30 mL)was added and the reaction mixture was stirred at room temperature foradditional 2 hours. The resulting solution was extracted with ethylacetate. The aqueous layer was concentrated in vacuo to approximately 20mL, Then concentrated NH₄OH (20 ml) was added and stirred overnight atroom temperature. The resulting mixture was concentrated under vacuumand the residue was diluted with 5 ml of water. The crude mixture wasthen purified with anion exchange chromatography on DEAE-Sephadex A-25at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude productwas further purified by reverse-phase HPLC to afford A5, which wascharacterized by MALDI-TOF MS calc'd for C₁₅H₂₆N₅O₁₂P₃S₂: 625.4, found:625.0.

Synthesis of 3′-O-tert-butyldithiomethyl-dCTP (Scheme 33)

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C2):To a solution ofN⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine (C1, 1.5 g, 3.4mmol) in DMSO (6.5 mL) with stirring was added acetic acid (2.91 mL) andacetic anhydride (9.29 mL). The reaction mixture was stirred at roomtemperature until the reaction for 2 days. Then the reaction mixture wasadded dropwise to solution of sodium bicarbonate and extracted by ethylacetate(50 ml×3). The obtained crude product was purified by columnchromatography (Ethyl Acetate/Hexane: 8/2) to give pure product 5 (1.26g, 74%) as a white solid. 1H NMR (400 MHz, CDCl₃) δ 8.43 (d, J=7.4 Hz,1H), 7.92 (d, J=7.6 Hz, 2H), 7.69-7.50 (m, 4H), 6.31 (t, J=6.1 Hz, 1H),4.75-4.59 (m, 2H), 4.51 (dt, J=6.2, 3.9 Hz, 1H), 4.20 (dt, J=3.7, 2.6Hz, 1H), 4.01 (dd, J=11.4, 2.9 Hz, 1H), 3.86 (dd, J=11.4, 2.4 Hz, 1H),2.72 (ddd, J=13.8, 6.2, 4.1 Hz, 1H), 2.18 (s, 4H), 0.97 (s, 9H), 0.17(d, J=3.9 Hz, 6H). HRMS (ESI⁺) calc'd for C₂₄H₃₅N₃O₅SSi[(M+H)⁺]:506.2145, found: 506.2146.

N⁴-Benzoyl-3′-O-tert-butyldithiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C3):N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C2, 1.01 g, 2 mmol) was dissolved in anhydrous dichloromethane (8 mL),followed by addition of triethylamine (278 μL, 2 mmol) and molecularsieves (3 Å, 1 g). The mixture was cooled in an ice-bath after stirringat room temperature for 0.5 hour and then a solution of sulfurylchloride (161 μL, 2.2 mmol) in anhydrous dichloromethane (8 mL) wasadded dropwise. The ice-bath was removed and the reaction mixture wasstirred further for 0.5 hour. Then potassium p-toluenethiosulfonate (678mg, 3 mmol) in anhydrous DMF (1 mL) was added to the mixture. Stirringwas continued at room temperature for additional 1 hour followed byaddition of tert-butyl mercaptan (1 mL). The reaction mixture wasstirred at room temperature for 0.5 hour and quickly filtered. Thesolvent was removed under reduced pressure and the residue was dissolvedin ethyl acetate and washed by brine(3×50 mL). The combined organiclayers were dried over Na₂SO₄ and filtered. The filtrate wasconcentrated to dryness under reduced pressure and the residue of thedesired compound was purified by silica gel column chromatography usinga gradient of ethyl acetate-Hexane from 3:7 (v/v) to 5:5 (v/v), yielding959 mg C3 as a white foam (83%). ¹H NMR (400 MHz, CDCl₃) δ 8.43 (d,J=7.4 Hz, 1H), 7.92 (d, J=7.6 Hz, 2H), 7.69-7.50 (m, 4H), 6.31 (t, J=6.1Hz, 1H), 4.75-4.59 (m, 2H), 4.51 (dt, J=6.2, 3.9 Hz, 1H), 4.20 (dt,J=3.7, 2.6 Hz, 1H), 4.01 (dd, J=11.4, 2.9 Hz, 1H), 3.86 (dd, J=11.4, 2.4Hz, 1H), 2.72 (ddd, J=13.8, 6.2, 4.1 Hz, 1H), 2.18 (s, 4H), 0.97 (s,9H), 0.17 (d, J=3.9 Hz, 6H), 0.10 (s, 2H). HRMS (ESI⁺) calc'd for:C₂₇H₄₁N₃O₅S₂Si [(M+Na)⁺]:602.2155, found: 602.2147.

N⁴-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxycytidine (C4): To astirred solution ofN⁴-Benzoyl-3′-O-tert-butyldithiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C3, 958 mg, 1.66 mmol) in a mixture of Tetrahydrofuran (24 ml),tetrabutylammonium fluoride (1.0M, 2.48 mL) was added in small portion,stirred at room temperature for 3 hours. The reaction mixture was pouredinto saturated sodium bicarbonate solution (50 mL) and extracted byEthyl Acetate (3×50 mL). The combined organic layers were dried overNa₂SO₄ and filtered. The filtrate was concentrated to dryness underreduced pressure and the residue of the desired compound was purified bysilica gel column chromatography using a gradient of ethylacetate-Hexane from 5:5 (v/v), affording 435 mg C4 as white solid powder(56%). ¹H NMR (400 MHz, Methanol-d₄) δ 8.52 (d, J=7.5 Hz, 1H), 8.04-7.96(m, 2H), 7.71-7.60 (m, 2H), 7.61-7.51 (m, 2H), 6.28-6.19 (m, 1H),4.95-4.86 (m, 2H), 4.54 (dt. J=6.0, 3.0 Hz, 1H), 4.23 (q, J=3.4 Hz, 1H),3.92-3.76 (m, 2H), 2.70 (ddd, J=13.9, 6.0, 2.9 Hz, 1H), 2.25 (ddd,J=13.6, 7.2, 6.2 Hz, 1H), 1.37 (s, 9H). HRMS (ESI⁺) calc'd forC₂₁H₂₇N₃O₅S₂[(M+Na)⁺]: 488.1290, found: 488.1297.

3′-O-tert-butyldithiomethyl-dCTP (C5):N⁴-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxycytidine (C4, 50 mg, 0.11mmol), tetrabutylammonium pyrophosphate (180 mg, 0.33 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).This mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 reaction mixture was added to the solutionof N⁴-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxycytidine and stirredfurther for 1 hour at room temperature. Iodine solution (0.02 Miodine/pyridine/water) was then injected into the reaction mixture untila permanent brown color was observed. After 10 min. water (30 mL) wasadded and the reaction mixture was stirred at room temperature foradditional 2 hours. The resulting solution was extracted with ethylacetate. The aqueous layer was concentrated in vacuo to approximately 20mL, Then concentrated NH₄OH (20 ml) was added and stirred overnight atroom temperature. The resulting mixture was concentrated under vacuumand the residue was diluted with 5 ml of water. The crude mixture wasthen purified with anion exchange chromatography on DEAE-Sephadex A-25at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude productwas further purified by reverse-phase HPLC to afford C5. HRMS (ESI−)calc'd for C₁₄H₂₅N₃O₁₃P₃S₂[(M−H)⁻]: 600.0042, found: 600.0033.

The Synthesis of 3′-O-Bodipy-DTM-dTTP and 3′-O-Bodipy-PEG₄-DTM-dTTP(Scheme 34)

3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl thymidine (T2): To asolution of the 5′-O-tert-Butyldimethylsilyl thymidine (T1, 1.07 g, 3mmol) in DMSO (10 mL) with stirring was added acetic acid (2.6 mL) andacetic anhydride (8.6 mL). The reaction mixture was stirred at roomtemperature until the reaction was complete (48 h), which was monitoredby TLC. Then the mixture was added slowly to the solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the residue of the desired compound was purified by silicagel column chromatography (ethyl acetate/hexane: 1/2) to give pureproduct T2 (0.97 g, 74%). ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (s, 1H), 7.48(s, 1H), 6.28 (m, 1H), 4.62 (m, 2H), 4.46 (m, 1H), 4.10 (m, 1H),3.78-3.90 (m, 2H), 2.39 (m, 1H), 2.14 (s, 3H), 1.97 (m, 1H), 1.92 (s,3H), 0.93 (s, 9H), 0.13 (s, 3H); HRMS (Fab⁺) calc'd for C₁₈H₃₃N₂O₅SSi[(M+H)+]: 417.1879, found: 417.1890.

Compound T6:

3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl thymidine (T2, 625mg, 1.50 mmol) was dissolved in anhydrous dichloromethane (20 mL),followed by addition of triethylamine (0.3 mL) and molecular sieves (3Å, 2 g). The mixture was cooled in an ice-bath after stirring at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.12mL, 1.50 mmol) in anhydrous dichloromethane (3 mL) was added dropwiseduring 2 minutes. The ice-bath was removed and the reaction mixture wasstirred further for 0.5 hour. Then potassium p-toluenethiosulfonate(0.61 g, 2.25 mmol) in anhydrous DMF (3 mL) was added to the mixture.Stirring was continued at room temperature for additional 1 hourfollowed by addition of2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide (403 mg, 2.01mmol). The reaction mixture was stirred at room temperature for 0.5 hourand quickly filtered through celite. The filter was washed withdichloromethane and the organic fraction was concentrated to give thecrude compound T6.

Compound T7:

Without the isolation, the crude compound T6 was dissolved in THF (10mL) followed by the addition of tetrabutylammonium fluoride THF solution(1.0M, 1.0 mL, 1.0 mmol). The mixture was stirred at room temperatureuntil the reaction was complete, which was monitored by TLC. Then, themixture was concentrated in vacuo, saturated NaHCO₃ solution (50 mL) wasadded and the mixture was extracted with dichloromethane. The organiclayer was dried over anhydrous Na₂SO₄, filtered, concentrated and theobtained crude mixture was purified by flash column chromatography(dichloromethane/methanol: 20/1) to give compound T7 (199 mg, 27% fromcompound T2). ¹H NMR (400 MHz, CDCl₃) δ 9.41 (s, 1H), 7.44 (s, 1H), 7.07(t, J=6.6 Hz, 1H), 6.11 (t, J=7.0 Hz, 1H), 4.88-4.80 (m, 2H), 4.57 (m,1H), 4.14 (q, J=2.9 Hz, 1H), 3.93 (m, 1H), 3.82 (m, 1H), 3.49 (d, J=6.2Hz, 2H), 3.10 (t, J=6.2, 4.1 Hz, 1H), 2.42-2.39 (m, 2H), 1.91 (s, 3H),1.31 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 164.39, 158.22, 150.95, 137.33,111.61, 87.33, 85.30, 80.39, 78.65, 77.66, 62.84, 50.70, 48.24, 37.28,25.74, 12.86: MS (APCI⁺) calc'd for CI₁H₂₄F₃N₃O₆S₂: 487.51. found:487.6.

3′-O—NH₂-DTM-dTTP (T8):

Compound T7 (50 mg, 103 pmol), tetrabutylammonium pyrophosphate (150 mg,0.27 mmol) and 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (33 mg,0.17 mmol) were dried separately over night under high vacuum at ambienttemperature. The tetrabutylammonium pyrophosphate was dissolved indimethylformamide (DMF, 1 mL) under argon followed by addition oftributylamine (1.5 mL). The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of 3′-O-t-Butyldithiomethyl thymidine and stirred further for 1hour at room temperature. Iodine solution (0.02 M iodine/pyridine/water)was then injected into the reaction mixture until a permanent browncolor was observed. After 10 min, Water (30 mL) was added and thereaction mixture was stirred at room temperature for additional 2 hour.The resulting solution was extracted with ethyl acetate. Thenconcentrated NH₄OH (20 ml) was added and stirred overnight at roomtemperature. The aqueous layer was concentrated in vacuo and the residuewas diluted with 5 ml of water. The crude mixture was then purified withanion exchange chromatography on DEAE-Sephadex A-25 at 4° C. using agradient of TEAB (pH 8.0; 0.1-1.0 M). The crude product was furtherpurified by reverse-phase HPLC to afford T8, which was characterized byMALDI-TOF MS, calc'd for C₁₅H₂N₃O₁₄P₃S₂: 631.45. found: 631.0.

3′-O-Bodipy-DTM-dTTP (compound T9):

To a stirred solution of Bodipy FL-NHS ester (1.5 mg, 3.9 pmol) in DMF(0.2 ml), 3′-O-DTM-dTTP (compound T8, 4.0 mmol) in NaHCO₃/Na₂CO₃ buffer(pH 8.9, 0.1 M, 0.3 ml). The reaction mixture was stirred at roomtemperature for 3 h with exclusion of light. The reaction mixture waspurified by a preparative silica gel TLC plate(dichloromethane/methanol, 4:1). The crude product was then purifiedwith anion exchange chromatography on DEAE-Sephadex A-25 at 4° C. usinga gradient of TEAB (pH 8.0; 0.1-1.0 M), and further purified onreverse-phase HPLC to afford 3′-O-DTM-Bodipy-dTTP 79, which wascharacterized by MALDI-TOF MS, calc'd for C₂₉H₄₁BF₂N₅O₁₅P₃S₂: 905.5,found: 904.1.

3′-O-Bodipy-PEG₄-DTM-dTTP (compound T10):

To a stirred solution of Bodipy-PEG₄-Acid (2.1 mg, 3.8 pmol) in dry DMF(200 μl), N,N-disuccinimidyl carbonate (1.03 mg, 4.0 pmol) and4-dimethylaminopyridine (0.48 mg, 4.0 pmol) were added. The reactionmixture was stirred at room temperature for 2 h. TLC indicated thatBodipy-PEG₄-Acid was completely converted to compound Bodipy-PEG₄-NHSester, which was directly used to couple with amino-3′-O-DTM-dTTP (3.8pmol) in NaHCO₃/Na₂CO₃ buffer (pH 8.9, 0.1 M) (300 μl). The reactionmixture was stirred at room temperature for 3 h with exclusion of light.The reaction mixture was purified by a preparative silica gel TLC plate(dichloromethane/methanol, 4:1). The crude mixture was purified withanion exchange chromatography on DEAE-Sephadex A-25 at 4° C. using agradient of TEAB (pH 8.0; 0.1-1.0 M), and further purified onreverse-phase HPLC to afford 3′-O-DTM-PEG₄-Bodipy-dTTP T10, which wascharacterized by MALDI-TOF MS calc'd for C₄₀H₆₂BF₂N₆O₂₀P₃S₂: 1152.8,found: 1151.4.

The Synthesis of 3′-O-Rox-DTM-dATP and 3′-O-Rox-PEG₄-DTM-dATP (Scheme35)

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine(A2): To a solution of theN⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine (A1, 1.41 g, 3mmol) in DMSO (10 mL) with stirring was added acetic acid (3 mL) andacetic anhydride (9 mL). The reaction mixture was stirred at roomtemperature until the reaction was complete (48 h), which was monitoredby TLC. Then the mixture was added slowly to the solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3>30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the residue of the desired compound was purified by silicagel column chromatography (dichloromethane/methanol: 30/1) to give pureproduct A2 (1.39 g, 88%). ¹H NMR (400 MHz, CDCl₃) δ 9.12 (s, 1H), 8.81(s, 1H), 8.35 (s, 1H), 8.10-8.01 (m, 2H), 7.68 (m, 1H), 7.49 (m, 2H),6.53 (dd, J=7.5, 6.0 Hz, 1H), 4.78-4.65 (m, 3H), 4.24 (dt, J=4.3, 3.1Hz, 1H), 3.98-3.81 (m, 2H), 2.80-2.60 (m, 2H), 2.21 (s, 3H), 0.94 (s,10H), 0.13 (s, 6H); MS (APCI⁺) calc'd for C₂₅H₃₅N₅O₄SSi: 529.73. found:529.4.

Compound A6:

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine(A2, 550 mg, 1.04 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.3 mL) and molecular sieves(3 Å, 2 g). The mixture was cooled in an ice-bath after stirring at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.12mL, 1.50 mmol) in anhydrous dichloromethane (3 mL) was added dropwiseduring 2 minutes. The ice-bath was removed and the reaction mixture wasstirred further for 0.5 hour. Then potassium p-toluenethiosulfonate(0.61 g, 2.25 mmol) in anhydrous DMF (3 mL) was added to the mixture.Stirring was continued at room temperature for additional 1 hourfollowed by addition of2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide (302 mg, 1.5mmol). The reaction mixture was stirred at room temperature for 0.5 hourand quickly filtered through celite. The filter was washed withdichloromethane and the organic fraction was concentrated to give thecrude compound A6: MS (APCI⁺) calc'd for C₃₀H₄₁F₃N₆O₅S₂Si: 714.89,found: 714.6.

Compound A7:

Without the isolation, the crude compound A6 was dissolved in THF (10mL) followed by the addition of tetrabutylammonium fluoride THF solution(1.0M, 1.0 mL, 1.0 mmol). The mixture was stirring at room temperatureuntil the reaction was complete, which was monitored by TLC. Then, themixture was concentrated in vacuo, saturated NaHCO₃ solution (50 mL) wasadded and the mixture was extracted with dichloromethane. The organiclayer was dried over anhydrous Na₂SO₄, filtered, concentrated and theobtained crude mixture was purified by flash column chromatography(dichloromethane/methanol: 20/1) to give compound A7 (128 mg, 20% fromcompound A2). ¹H NMR (400 MHz, CDCl₃) δ 9.16 (s, 1H), 8.77 (s, 1H), 8.11(s, 1H), 8.07-8.00 (m, 2H), 7.61 (m, 1H), 7.56-7.52 (m, 2H), 6.91 (m,1H), 6.33 (dd, J=9.4, 5.5 Hz, 1H), 5.83 (d, J=10.7 Hz, 1H), 4.88 (d,J=2.6 Hz, 2H), 4.75 (dt, J=5.4, 1.2 Hz, 1H), 4.36 (q, J=1.7 Hz, 1H),4.03 (dd, J=12.8, 1.8 Hz, 1H), 3.81 (t, J=10.9 Hz, 1H), 3.51 (d, J=6.2Hz, 2H), 3.10 (m, 1H), 2.56-2.46 (m, 1H), 1.36 (s, 6H); ¹³C NMR (75 MHz,CDCl₃) δ 164.91, 152.49, 151.03, 150.71, 142.95, 133.82, 133.33, 129.29,128.29, 125.00, 118.23, 114.41, 88.01, 87.10, 80.37, 80.19, 63.91,60.76, 50.66, 47.99, 38.17, 25.82, 25.75; MS (APCI⁺) calc'd forC₂₄H₂₇F₃N₆O₅S₂: 600.6, found: 600.7.

3′-NH₂-DTM-dATP (A8:

Compound A7 (50 mg, 103 pmol), tetrabutylammonium pyrophosphate (150 mg,0.27 mmol) and 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (33 mg,0.17 mmol) were dried separately over night under high vacuum at ambienttemperature. The tetrabutylammonium pyrophosphate was dissolved indimethylformamide (DMF, 1 mL) under argon followed by addition oftributylamine (1.5 mL). The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of 3′-O-t-Butyldithiomethyl thymidine and stirred further for 1hour at room temperature. Iodine solution (0.02 M iodine/pyridine/water)was then injected into the reaction mixture until a permanent browncolor was observed. After 10 min, Water (30 mL) was added and thereaction mixture was stirred at room temperature for additional 2 hour.Then concentrated NH₄OH (20 ml) was added and stirred overnight at roomtemperature. The resulting solution was extracted with ethyl acetate.The aqueous layer was concentrated in vacuo to approximately 20 mL, Thenconcentrated NH₄OH (20 ml) was added and stirred overnight at roomtemperature. The resulting mixture was concentrated under vacuum and theresidue was diluted with 5 ml of water. The crude mixture was thenpurified with anion exchange chromatography on DEAE-Sephadex A-25 at 4°C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude product wasfurther purified by reverse-phase HPLC to afford A8, which wascharacterized by MALDI-TOF MS, calc'd for C₁₅H₂₇N₆O₁₂P₃S₂: 640.45.found: 639.6.

3′-O-Rox-DTM-dATP (compound A9):

To a stirred solution of ROX-NHS ester (2 mg, 3.2 pmol) in DMF (0.2 ml),amino 3′-O-DTM-dATP (compound A8, 3.0 pmol) in NaHCO₃/Na₂CO₃ buffer (pH8.9, 0.1 M, 0.3 ml). The reaction mixture was stirred at roomtemperature for 3 h with exclusion of light. The reaction mixture waspurified by a preparative silica gel TLC plate(dichloromethane/methanol, 4:1). The crude product was then purifiedwith anion exchange chromatography on DEAE-Sephadex A-25 at 4° C. usinga gradient of TEAB (pH 8.0; 0.1-1.0 M), and further purified onreverse-phase HPLC to afford 3′-O-DTM-Rox-dATP A9, which wascharacterized by MALDI-TOF MS, calc'd for C₄₈H₅₅N₈O₁₆P₃S₂: 1157.0,found: 1155.4.

3′-O-Rox-PEG₄-DTM-dATP (compound A 10):

To a stirred solution of ROX-PEG₄-Acid (2.6 mg, 3.3 pmol) in dry DMF(200 μl), N,N-disuccinimidyl carbonate (0.90 mg, 3.5 pmol) and4-dimethylaminopyridine (0.43 mg, 3.5 pmol) were added. The reactionmixture was stirred at room temperature for 2 h. TLC indicated thatROX-PEG₄-Acid was completely converted to compound ROX-PEG₄-NHS ester,which was directly used to couple with amino-3′-O-DTM-dATP (3.5 pmol) inNaHCO₃/Na₂CO₃ buffer (pH 8.9, 0.1 M) (300 μl). The reaction mixture wasstirred at room temperature for 3 h with exclusion of light. Thereaction mixture was purified by a preparative silica gel TLC plate(dichloromethane/methanol, 4:1). The crude mixture was then purifiedwith anion exchange chromatography on DEAE-Sephadex A-25 at 4° C. usinga gradient of TEAB (pH 8.0; 0.1-1.0 M), and further purified onreverse-phase HPLC to afford 3′-O-DTM-PEG₄-Rox-dATP A10, which wascharacterized by MALDI-TOF MS, calc'd for C₅₉H₇₆N₉O₂₁P₃S₂: 1404.3,found: 1401.6.

Synthesis of 3′-O-TCO-DTM-dTTP (Scheme 36): Compound T8 (1 mg, 1.6 μmol)was dissolved in 0.1 M NaHCO₃/Na₂CO₃ (500 μL, pH=8.8), followed byaddition of trans-cyclooctenyl NHS ester (1 mg, 3.7 μmol) in anhydrideDMF (500 μL), stirring at r.t. for 4 hours. The product was purified byreverse-phase HPLC to give pure T11, which was characterized by HRMS,calc'd for C₂₁H₃₃N₆O₁₆P₃ [M−H]: 717.1088, found: 717.1100.

Synthesis of 3′-O-Biotin-DTM-dCTP (Scheme 37)

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C2): To a solution of theN⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine (C1, 2.25 g,2.51 mmol) in DMSO (20 mL) with stirring was added acetic acid (8 mL)and acetic anhydride (20 mL). The reaction mixture was stirred at roomtemperature until the reaction was complete (24 h), which was monitoredby TLC. Then the mixture was added slowly to the solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×50 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the residue of the desired compound was purified by silicagel column chromatography (DCM/MeOH: 1/30) to give pure product C2 (2.13g, 83%). ¹H NMR (400 MHz. CDCl₃) δ: 8.43 (d, J=8.4 Hz, 1H), 7.92 (d,J=7.6 Hz, 2H), 7.66 (m, 1H), 7.53 (m, 3H), 6.30 (t, J=6.0 Hz, 1H), 4.69(dd, J=32 Hz, 7.6 Hz, 2H), 4.50 (m, 1H), 4.18 (m, 1H), 3.98-3.83 (m,2H), 2.74 (m, 1H), 2.21-2.12 (m, 4H), 0.93 (s, 9H), 0.15 (m, 6H). MS(APCI⁺) calc'd for C₂₄H₃₅N₃O₅SSi: 505.70, found: 505.6.

Compound C6:

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C2, 0.87 mg, 1.72 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.3 mL) and molecular sieves(3 Å, 2 g). The mixture was cooled in an ice-bath after stirring at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.15mL, 1.80 mmol) in anhydrous dichloromethane (3 mL) was added dropwiseduring 2 minutes. The ice-bath was removed and the reaction mixture wasstirred further for 0.5 hour. Then potassium p-toluenethiosulfonate(0.68 g, 2.5 mmol) in anhydrous DMF (3 mL) was added to the mixture.Stirring was continued at room temperature for additional 1 hourfollowed by addition of2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide (402 mg, 2.0mmol). The reaction mixture was stirred at room temperature for 0.5 hourand quickly filtered through celite. The filter was washed withdichloromethane and the organic fraction was concentrated to give thecrude compound C6: MS (APCI) calc'd for C₂₉H₄₁F₃N₄O₆S₂Si: 690.87, found:689.8 [M−H]⁻.

Compound C7:

Without the isolation, the crude compound C6 was dissolved in THF (10mL) followed by the addition of tetrabutylammonium fluoride THF solution(1.0M, 1.0 mL, 1.0 mmol). The mixture was stirring at room temperatureuntil the reaction was complete, which was monitored by TLC. Then, themixture was concentrated in vacuo, saturated NaHCO₃ solution (50 mL) wasadded and the mixture was extracted with dichloromethane. The organiclayer was dried over anhydrous Na₂SO₄, filtered, concentrated and theobtained crude mixture was purified by flash column chromatography(dichloromethane/methanol: 20/1) to give compound C7 (171 mg, 17% fromcompound C2). ¹H NMR (400 MHz, CDCl₃) δ 8.94 (br, 1H), 8.32 (d, J=7.5Hz, 1H), 7.90-7.83 (m, 2H), 7.65-7.55 (m, 2H), 7.49 (dd, J=8.4, 7.1 Hz,2H), 7.12 (t, J=6.3 Hz, 1H), 6.15 (t, J=6.4 Hz, 1H), 4.93-4.78 (m, 2H),4.58 (dt, J=6.5, 3.3 Hz, 1H), 4.24 (q, J=3.0 Hz, 1H), 4.02 (dd, J=12.1,3.0 Hz, 1H), 3.86 (dd, J=12.1, 2.9 Hz, 1H), 3.66 (br, 1H), 3.50 (d,J=6.2 Hz, 2H), 2.71 (m, 1H), 2.40 (m, 1H), 1.34 (s, 6H). ¹³C NMR (75MHz, CDCl₃) δ 162.82, 158.20, 157.72, 155.45, 146.01, 133.63, 129.40,127.98, 97.24, 89.14, 86.04, 80.46, 78.25, 62.47, 50.72, 48.04, 38.47,25.78; MS (APCI⁺) calc'd for C₂₃H₂₇F₃N₄O₆S₂: 576.61. found: 576.

3′-NH₂-DTM-dCTP (C8):

Compound C7 (50 mg, 87 pmol), tetrabutylammonium pyrophosphate (140 mg,0.25 mmol) and 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (30 mg,0.15 mmol) were dried separately over night under high vacuum at ambienttemperature. The tetrabutylammonium pyrophosphate was dissolved indimethylformamide (DMF, 1 mL) under argon followed by addition oftributylamine (1.5 mL). The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of 3′-O-t-Butyldithiomethyl thymidine and stirred further for 1hour at room temperature. Iodine solution (0.02 M iodine/pyridine/water)was then injected into the reaction mixture until a permanent browncolor was observed. After 10 min, Water (30 mL) was added and thereaction mixture was stirred at room temperature for additional 2 hour.The resulting solution was extracted with ethyl acetate. The aqueouslayer was concentrated in vacuo and the residue was diluted with 5 ml ofwater. The crude mixture was then purified with anion exchangechromatography on DEAE-Sephadex A-25 at 4° C. using a gradient of TEAB(pH 8.0; 0.1-1.0 M), and further purified by reverse-phase HPLC toafford C8, which was characterized by MALDI-TOF MS, calc'd forC₁₄H₂₉N₄O₁₃P₃S₂: 618.4. found: 616.7.

3′-Biotin-DTM-dCTP (C9): To a stirred solution of Bio-NHS ester (2 mg,3.2 pmol) in DMF (0.2 ml), amino 3′-O-DTM-dCTP (compound C8, 3.0 pmol)in NaHCO₃/Na₂CO₃ buffer (pH 8.9, 0.1 M, 0.3 ml). The reaction mixturewas stirred at room temperature for 3 h with exclusion of light. Thecrude product was then purified with anion exchange chromatography onDEAE-Sephadex A-25 at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0M), and further purified on reverse-phase HPLC to afford3′-O-Biotin-DTM-dATP C9, which was characterized by MALDI-TOF MS,calc'd: 842, found: 842.5.

The syntheses of 3′-O-DTM-Anchor-SS(DTM)-dNTPs (structures shown in FIG.4) are described in Schemes below.

Synthesis of Dye Labeled Binding Molecules

Synthesis of Labeled Binding Molecules Conjugated with Fluorescent Dyesis conducted by coupling commercially available binding moleculestarting materials with various activated Dyes. Example synthesis of RoxLabeled Tetrazine, Alexa488 Labeled SHA and R6G LabeledDibenzocyclooctyne(DBCO) is shown in Scheme 42.

Synthesis of multiple-dye conjugated binding molecules (Cy5-tetrazine asan example) is shown in Schemes 43-45.

Synthesis of Rox-7-Cy5 Labeled SHA (Shown FIG. 10A)

Cy5 labeled CPG (Glen Research) is used to start solid phaseoligonucleotide synthesis on a DNA synthesizer, dSpacer phosphoramiditeis used as the building block for seven consecutive coupling cycles,then Rox labeled dT phosphoramidite is used in the next coupling cycle.C5 amino modifier phosphoramidite is used in the last coupling cycle.After cleavage under mild condition following the GlenResearch protocol,the amino modified Rox-7-Cy5 product is produced and purified by HPLC.Coupling of SHA NHS ester with amino modified Rox-7-Cy5 in DMSO/NaCO₃,NaHCO₃ buffer (pH 8.9) will afford Rox-7-Cy5 labeled SHA.

Synthesis of Rox-3-Cy5 Labeled DBCO (Shown in FIG. 10 B)

Cy5 labeled CPG (Glen Research) is used to start solid phaseoligonucleotide synthesis on a DNA synthesizer, dSpacer phosphoramiditeis used as the building block for three consecutive coupling cycles,then Rox labeled dT phosphoramidite is used in the next coupling cycle.C5 amino modifier phosphoramidite is used in the last coupling cycle.After cleavage under mild condition following the GlenResearch protocol,the amino modified Rox-3-Cy5 product is produced and purified by HPLC.Coupling of DBCO NHS ester with amino modified Rox-3-Cy5 in DMSO/NaCO₃,NaHCO₃ buffer (pH 8.9) will afford Rox-3-Cy5 labeled DBCO.

Syntheses of Labeled Binding Molecules Conjugated with Fluorescent Dyevia Different Cleavable Linkers (the structures of these molecules areshown in FIG. 12) are shown in Schemes 46 to 52.

Synthesis of Labeled Binding Molecules Conjugated with Fluorescent Dyesis achieved by coupling commercially available activated Dyes withbinding molecules containing cleavable linkage moieties, which aresynthesized using commercially available materials.

The example synthesis of SHA-2-Nitrobenzyl (linker)-ATTO647N is shown inScheme 46; The example synthesis of Tetrazine-Azo(linker)-ATTO647N isshown in Scheme 47 and the construction of the Azo linker moiety isaccomplished using literature method;⁴¹ The example synthesis ofStreptavidin-Dimethylketal(linker)-ATTO647N is shown in Scheme 48 andthe construction of the Dimethylketal linker moiety is accomplishedusing literature method;⁴² The example synthesis ofDibenzocyclooctyne(DBCO)-Allyl(linker)-ATTO647N is shown in Scheme 49:The example synthesis of Dibenzocyclooctyne(DBCO)-Dde(linker)-ATTO647Nis shown in Scheme 50 and the construction of the Dde linker moiety isaccomplished using literature method.⁴³ The example synthesis ofTerazine-Dde(linker)-ATTO647N and Terazine-Dde(linker)-ROX is shown inScheme 51: The example synthesis of DBCO-Azo(—N═N-Linker)-ATTO647N andDBCO-Azo(—N═N-Linker)-ROX is shown in Scheme 52.

Detailed cleavage reaction and the cleaved products using linkersconstructed from Azo, Dimethylketal and Dde under mild conditions (usingN₂S₂O₄, Citric acid and N₂H₄ respectively) are shown in Scheme 55 usingTetrazine-Azo(linker)-ATTO647N,Streptavidin-Dimethylketal(linker)-ATTO647N) andDibenzocyclooctyne-Dde(linker)-ATTO647N described above as examples.

Example Synthesis of 3′-O-Rox-Nitrobenzyl-dCTP and 3′-O-Rox-Allyl-dTTPare shown in Scheme 53 and Scheme 54 respectively.

Consecutive Polymerase Extension using 3′-O-Rox-DTM-dATP ReversibleTerminator and Characterization by MALDI-TOF Mass Spectrometry (Resultsare shown in FIGS. 34A-34C)

This extension reaction was carried out using 200 pmol of reversibleterminator (3′-O-Rox-DTM-dATP), 2 units of Therminator™ IX DNAPolymerase (A 9N™ DNA Polymerase variant from NEB), 100 pmol of DNAprimer (5′-TAGATGACCCTGCCTTGTCG-3′) (SEQ ID NO: 18), 60 pmol of DNAtemplate (5′-GAAGGAGACACGCGGCCAGAGAGGTCCTGTCCCGTGTCCGTGTGTGCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTTTTCTCTTCGTTCTC CGT-3′) (SEQID NO:19) in a 20 μl buffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄,10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH 8.8 @ 25° C., and 2 mMMnCl₂. The reaction was conducted in an ABI GeneAmp PCR System 9700 withinitial incubation at 65° C. for 30 second, followed by 38 cycles of 65°C./30 sec, 45° C./30 sec, 65° C./30 sec. Multiple reactions were carriedout and an aliquot of the reaction mixture was desalted using a C18ZipTip column (Millipore, MA) and analyzed by MALDI-TOF MS (ABI VoyagerDE).

Calf Intestinal Alkaline Phosphatase (CIP) from NEB was used toinactivate residual reversible terminator nucleotide and THP(Tris-hydroxypropyl-phosphine) was used to remove the Rox-tBu-SS groupfrom the 3′ end of the DNA extension product to regenerate the 3′-OHgroup in preparation for the next extension reaction. The cleavagereaction was carried out by incubating the extension reaction mixturewith THP at 5 mM final concentration and incubating at 65° C. for 5minutes.

The reaction mixture after THP treatment was purified by reverse phaseHPLC on an XTerra MS C18, 2.5 μm 4.6 mm×50 mm column (Waters, MA) toobtain the pure cleavage product. Mobile phase: A, 8.6 mMtriethylamine/100 mM 1,1,1,3,3,3-hexafluoro-2-propanol in water (pH8.1); B, methanol. Elution was performed at 40° C. with a 0.5 mL/minflow rate with a linear gradient from 88% A/12% B to 65.5% A/34.5% B for90 min. The purified product was used in the subsequent extensionreaction.

Since there are two consecutive Ts on the DNA template after the DNAprimer binding site, the second extension reaction was carried out inthe same way as the first extension reaction. The overall results areshown in FIGS. 34A-34C, MALDI TOF MS of the primers after each stepdemonstrate accurate incorporation of 3′-Rox-SS-dATP, efficient cleavageof SS bond and recovering of 3′OH, and incorporation of another3′-Rox-SS-dATP.

DNA Polymerase Extension using 3′-O-Rox-PEG₄-DTM-dATP ReversibleTerminator, cleavage reaction using THP, and characterization byMALDI-TOF Mass Spectrometry (Results are shown in FIGS. 35A-35C)

The DNA Polymerase extension was carried out using 200 pmol ofreversible terminator (3′-O-Rox-PEG₄-DTM-dATP), 2 units of Terminator™IX DNA Polymerase (NEB), 100 pmol of primer (5′-TAGATGACCCTGCCTTGTCG-3′)(SEQ ID NO:20), 60 pmol of DNA template(5′-GAAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTTGTGCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTTTTCTCTTCGTTCTCCGT-3′) (SEQ ID NO:21)in a 20 μl buffer containing 20 mM Tris-HCl, 10 mM (NH)₂SO₄, 10 mM KCl,2 mM MgSO₄, 0.1% Triton X-100, pH 8.8 @ 25° C., and 2 mM MnCl₂. Thereaction was conducted in an ABI GeneAmp PCR System 9700 with initialincubation at 65° C. for 30 second, followed by 38 cycles of 65° C./30sec, 45° C./30 sec, 65° C./30 sec. The reaction mixture was thendesalted using a C18 ZipTip column (Millipore, MA) and analyzed byMALDI-TOF MS (ABI Voyager DE).

Calf Intestinal Alkaline Phosphatase (CIP) from NEB was used toinactivate residual reversible terminator nucleotide and THP was used toremove the blocking group from the 3′ end of the DNA extension productto regenerate the 3′-OH group. The cleavage reaction was carried out byincubating the extension reaction mixture with THP at 5 mM finalconcentration and incubating at 65° C. for 5 minutes.

DNA Polymerase Extension using either 3′-O-Bodipy-DTM-dTTP, or3′-O-Bodipy-PEG₄-DTM-dTTP Reversible Terminator, cleavage reaction usingTHP, and characterization by MALDI-TOF Mass Spectrometry (Results areshown in FIGS. 10A-10D and FIG. 11)

The DNA Polymerase extension was carried out using 200 pmol ofreversible terminator (3′-O-Bodipy-DTM-dTTP, or3′-O-Bodipy-PEG4-DTM-dTTP), 2 units of Terminator™ IX DNA Polymerase(NEB), 100 pmol of primer (5′-GATAGGACTCATCACCA-3′), (SEQ ID NO:22) 60pmol of DNA template(5′-GAAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTITTGTGCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTITTCTCTTCGTCTCCGT-3′) (SEQ ID NO:23)in a 20 μl buffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl,2 mM MgSO₄, 0.1% Triton X-100, pH 8.8 @ 25° C., and 2 mM MnCl₂. Thereaction was conducted in an ABI GeneAmp PCR System 9700 with initialincubation at 65° C. for 30 second, followed by 38 cycles of 65° C./30sec, 45° C./30 sec, 65° C./30 sec. The reaction mixture was desaltedusing a C18 ZipTip column (Millipore, MA) and analyzed by MALDI-TOF MS(ABI Voyager DE).

Calf Intestinal Alkaline Phosphatase (CIP) from NEB was used toinactivate residual reversible terminator nucleotide in the extensionreaction mixture and THP was used to remove the blocking group from the3′ end of the DNA extension product to regenerate the 3′-OH group. Thecleavage reaction was carried out by incubating the extension reactionmixture with THP at 5 mM final concentration and incubating at 65° C.for 5 minutes. The reaction mixture was desalted using a C18 ZipTipcolumn (Millipore, MA) and analyzed by MALDI-TOF MS (ABI Voyager DE).

REFERENCES FOR EXAMPLE 2

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Ju J. et al.,“Four-color DNA sequencing by synthesis using cleavable fluorescentnucleotide reversible terminators”. Proc. Natl. Acad. Sci. USA 103(52);19635-19640 (2006).; 9. Wu J. et al., “3′-O-modified nucleotides asreversible terminators for pyrosequencing”, Proc. Natl. Acad. Sci. USA104(104); 16462-16467 (2007).; 10. Guo J. et al., “Four-color DNAsequencing with 3′-O-modified nucleotide reversible terminators andchemically cleavable fluorescent dideoxynucleotides”. Proc. Natl. Acad.Sci. USA 105(27); 9145-9150 (2008).; 11. Bentley D. R. et al., “Accuratewhole human genome sequencing using reversible terminator chemistry”,Nature 456(7218); 53-59 (2008).; 12. Harris T. D. et al.,“Single-molecule DNA sequencing of a viral genome”, Science 320(5872);106-109 (2008).; 13. Eid J. et al., “Real-time DNA sequencing fromsingle polymerase molecules”, Science 323(5910); 133-138 (2009).; 14.Rothberg J. M. et al., “An integrated semiconductor device enablingnon-optical genome sequencing”, Nature 475(7356); 348-352 (2011).; 15.Rosenblum B. B. et al., “New dye-labeled terminators for improved DNAsequencing patterns”, Nucleic Acids Res., 25, 4500-4504 (1997).; 16. ZhuZ. et al., “Directly labeled DNA probes using fluorescent nucleotideswith different length linkers”, Nucleic Acids Res., 22, 3418-3422(1994).; 17. Pelletier H. et al., “Structures of ternary complexes ofrat DNA polymerase beta, a DNA template-primer, and ddCTP”, Science,264, 189-1903 (1994).; 18. Cheeseman P., “Method for sequencingpolynucleotides”, U.S. Pat. No. 5,302,509 (1994).; 19. Canard B. et al.,“DNA polymerase fluorescent substrates with reversible 3′-tags”, Gene.,148, 1-6 (1994).; 20. Welch M. B. et al., “Synthesis of nucleosidesdesigned for combinatorial DNA sequencing”, Chem. Eur. J., 5, 951-960(1999).; 21. Metzker M. L., “Emerging technologies in DNA sequencing”,Genome Res., 15, 1767-1776 (2005).; 22. Lu G. et al., “A diversityoriented synthesis of 3′-O-modified nucleoside triphosphates for DNAsequencing by synthesis. Bioorg”, Med. Chem. Lett., 16, 3902-3905(2006).; 23. Kim T. S. et al., “Novel 3′-O-fluorescently modifiednucleotides for reversible termination of DNA synthesis”, ChemBiochem,11, 75-78 (2010).; 24. Kim D. R. et al., “Synthesis of3′-O-fluorescently mono-modified reversible terminators and their usesin sequencing-by-synthesis”. Bioorg. Med. Chem. Lett., 24, 209-213(2014).; 25. Hutter D. et al., “Labeled nucleoside triphosphates withreversibly terminating aminoalkoxy groups”, Nucleos. Nucleot. Nucl., 29,879-895 (2010).; 26. Diana C. et al., “Fluoride-Cleavable, FluorescentlyLabelled Reversible Terminators: Synthesis and Use in Primer Extension”,Chem. Eur. J., 17, 2903-2915 (2011).; 27. Kwiatkowski M., “Compounds forprotecting hydroxyls and methods for their use”, U.S. Pat. No. 7,279,563(2007).; 28. Muller S. et al., “Method for producing trinucleotides”,PCT International Application Publication No. WO 2011/061114 (2011).;29. Semenyuk A. et al., “Synthesis of RNA using 2′-O-DTM protection”, J.Am. Chem. Soc., 128, 12356-12357 (2006).; 30. John C. et al., “RapidCu-Free Click Chemistry with Readily SynthesizedBiarylazacyclooctynones”, J. Am. Chem. Soc., 132, 3688-3690 (2010).; 31.Shieha P. et al., “Design strategies for bioorthogonal smart probes”,Org. Biomol. Chem., 12, 9307-9320 (2014).; 32. Melissa L. et al.,“Tetrazine Ligation: Fast Bioconjugation Based onInverse-Electron-Demand Diels-Alder Reactivity”, J. Am. Chem. Soc., 130,13518-13519 (2008).; 33. Marjoke F. et al., “Bioorthogonal labelling ofbiomolecules; new functional handles and ligation methods”, Org. Biomol.Chem., 11, 6439-6455 (2013).; 34. Bergseid M. et al., “Smallmolecule-based chemical affinity system for the purification ofproteins”, BioTechniques, 29, 1126-1133 (2000).; 35. Anthony K. et al.,“Triple Fluorescence Energy Transfer in CovalentlyTrichromophore-Labeled DNA”, J. Am. Chem. Soc., 123, 12923-12924(2001).; 36. Ju J., “Sets of labeled energy transfer fluorescent primersand their use in multi component analysis”, U.S. Pat. No. 5,952,180(1999).; 37. Leriche G. et al., “Optimization of the Azobenzene Scaffoldfor Reductive Cleavage by Dithionite; Development of an AzobenzeneCleavable Linker for Proteomic Applications”, Eur. J. Org. Chem., 23,4360-64 (2010).; 38. Budin G. et al., “Nondenaturing Chemical Proteomicsfor Protein Complex Isolation and Identification” Chem Bio Chem, 11,2359-2361 (2010).; 39. Binaulda S. et al., “Acid-degradable polymers fordrug delivery; a decade of innovation”, Chem. Commun., 49, 2082-2102(2013).; 40. Ellis R. A. et al., “Chemical constructs”. European PatentNo. EP 1119529 B1 (2003).; 41. Rathod K. M. et al., “Synthesis andantimicrobial activity of azo compounds containing m-cresol moiety”,Chem. Sci. Tran., 2, 25-28 (2013).; 42. Shenoi R. A. et al., “BranchedMultifunctional Polyether Polyketals; Variation of Ketal Group StructureEnables Unprecedented Control over Polymer Degradation in Solution andwithin Cells”, J. Am. Chem. Soc., 134, 14945-14957 (2012).; 43. ChhabraS. R. et al., “An appraisal of new variants of Dde amine protectinggroup for solid phase peptide synthesis Tetra”. Lett., 39, 1603-1606(1998).

Example 3: Single-Molecule Electronic Sequencing by Synthesis Using3′-O-Anchor-Cleavable Linker Nucleotides/Polymer Tags and NanoporeDetection

Nanopore-based electronic single molecule real time DNA sequencing bysynthesis approaches have been previously developed (Kumar et alScientific Reports (2012) 2, 684; Fuller et al, PNAS USA (2016) 113,5233-5238). A nanopore SBS sequencing method that combines SBS withnanopore-based identification of different-sized polymer tags attachedto the terminal phosphate of the nucleotides has been reported. One offour different-length PEG tags was attached to the terminal phosphate ofeach nucleotide. Despite having long tags with 16-36 PEG monomer units,these tagged nucleotides were incorporated efficiently by DNApolymerase. During the phosphoryl transfer step of the DNA polymerasereaction, the tag is released as part of the polyphosphate byproduct, soonly the natural nucleotide remains in the growing DNA strand. This tagwas detected and identified by monitoring pore current as it passedthrough a single-protein nanopore (α-hemolysin) embedded in a lipidmembrane under a voltage gradient. Depending on the length of the PEGtag, the pore current was reduced to different levels, and translocationrequired different times (Kumar et al Scientific Reports (2012) 2, 684),allowing discrimination of such tags and enabling the identification ofeach nucleotide incorporated in the SBS process.

To develop this nanopore SBS approach further and to optimize the tags,Fuller et al (PNAS USA (2016) 113, 5233-5238) have reported the designand synthesis of nucleotides tagged with modified oligonucleotides andtheir application for nanopore SBS. These tags have structuralmodifications that create distinct ionic current blockades, measuredusing an electronic chip-based array of nanopores embedded in lipidbilayer membranes. The tags are attached to the terminal phosphate of2′-deoxynucleoside-5′-hexaphosphates using Huisgen cycloadditionazide/alkyne coupling chemistry (Fuller et al. US Patent ApplicationUS20150368710). With these tagged nucleotides, continuoussingle-molecule electronic DNA sequencing with single-base resolution bynanopore SBS was demonstrated. The measurement of current is made duringthe polymerase catalytic cycle when the complementary tagged nucleotideis bound within the complex of DNA polymerase, primer/template, anddivalent metal cation and lasts until the completion of the polymerasecatalytic step with the release of the tagged polyphosphate product.Once this product is released, the polymer tag is free to leave thepore, ending the blockade signal for that particular DNA synthesis step.To increase the likelihood that each tag will be measured in sequentialorder, a single polymerase molecule is covalently attached to thenanopore at an appropriate distance to allow fast capture of the tag bythe nanopore Each of the four tags has a distinctive structure thatinteracts with the narrowest constriction in the αHL channel, therebyreducing the ionic current across the channel to different extents(Fuller et al, PNAS USA (2016) 113, 5233-5238) In this approach, it iscritical to control the relative rates of the polymerase reaction, thecapture of tags by the nanopore, and the ionic current monitoring toensure that each and every base is called in sequential order. Failureto do so will result in “insertion” or “deletion” artifacts.

We reason that the above obstacles can be overcome by purposely pausingthe reactions by addition of a nucleotide with a cleavable 3′-OHblocking group (linker) containing an anchor to which a nanopore tagwith an anchor binding molecule can be attached. The followingelectronic detection of such a tag would result in one base at a timebeing called, even in homopolymeric tracts (runs of nucleotides with thesame base such as A_(n) or C_(n), where n>1). A few such 3′-O-cleavablelinkers that still allow incorporation of the nucleotides bearing suchlinkers into a growing DNA strand exist, including the 3′-O-dithiomethyllinker described herein.

3′-O-cleavable linker (dithiomethyl, DTM) nucleotides having an anchormoiety attached to the cleavable linker described in this applicationcan be used for such nanopore-based sequencing approaches. A bindingmolecule compatible with the anchor on the DTM linker would be attachedto a nanopore tag specific for each of the four nucleotides (A, C. G andT/U). Briefly, after incorporation into a primer of one of the3′-O-cleavable linker nucleotides bearing an anchor due to base pairingwith the complementary nucleotide on the template strand, labeling withthe nanopore tags containing anchor binding partners will reveal whichnucleotide was added at that step; subsequent cleavage of the linkerwill release the tags in preparation for the next incorporation. In thisprocess, the 3′-OH group will be restored so that the growing DNA strandwill bear only natural nucleotides.

Single-Molecule SBS by a Nanopore Using 3′-O-Anchor-Cleavable LinkerNucleotides (General Approach)

Thus, in an embodiment of the present invention, the nucleotide analoguecomprising a cleavable linker (DTM) at the 3′-O-position of thenucleotide is covalently linked to the anchor moiety (e.g. biotin,azide, trans-cyclooctene (TCO), phenylboronic acid (PBA), quadricyclane,norbornene). These anchor moieties can react in biorthogonal fashionwith their binding partner (e.g. streptavidin, dibenzylcyclooctene(DBCO), tetrazine, salicylhydroxamic acid (SHA),bis(dithiobenzil)nickel(II) compounds, nitrile oxide containingcompounds (Zheng et al, Molecules (2015) 20, 3190-3205; Springer et alJ. Biomol. Tech. (2003) 14, 183-190; Sletten and Bertozzi (2011) 133,17570; Gutsmiedl et al. Org Lett (2009) 11, 2405)). Some of the abovemolecules can be placed either on the 3′-position of the nucleotide asanchors or on the tag as binding molecules. For instance, PBA orphenyldiboronic acid (PDBA) reacts with SHA molecules to form a complexunder a variety of conditions; biotin complexes with streptavidin; theazido group reacts with DBCO; tetrazine reacts with trans-cycloocteneand norbornene in an efficient manner; quadricyclane complexes withbis(dithiobenzil)nickel(II) compounds, and norbornene conjugates withnitrile oxide (see examples in FIG. 37);

Wherein each of the at least four 3′-O-Anchor-DTM nucleotides comprisesa triphosphate or a polyphosphate, a base which is adenine, guanine,cytosine, thymine, or uracil, or a derivative of each thereof, and ananchor molecule covalently coupled to the 3′-O-position of thenucleotide sugar moiety comprising a cleavable linker (DTM) at the3′-O-position (examples in FIGS. 38, 39 and 42);

Wherein (i) the type of base in each anchor attached nucleotide isdifferent from the type of base in each of the other three anchor taggednucleotides, and (ii) determining which anchor nucleotide has beenincorporated into the primer to form a DNA extension product in thefirst step is accomplished by adding the 4 different tags tethered withdifferent binding partners which will either be covalently attached toor complexed with the corresponding anchor moieties attached via the3′-O-cleavable linker (DTM) moiety;

Wherein the anchor and or binding moiety is selected from azido,dibenzocyclooctyne, tetrazine, cyclooctene, norbornene, biotin, SHA,PBA, quadricyclane, nitrile oxide, bis(dithiobenzil)nickel(II)compounds, or streptavidin (FIG. 37);

Wherein the nanopore tag is an oligonucleotide, peptide, PEG,carbohydrate or a combination thereof (Fuller et al U.S. PatentApplication US20150368710) (examples in FIG. 40);

Wherein the nanopore tag is conjugated to the binding molecule (FIG.41), using synthetic schemes such as those shown in FIGS. 43-46.

Variants of Single-Molecule SBS by a Nanopore Using3′-O-Anchor-Cleavable Linker Nucleotides

In the following four embodiments, either polymerase (FIGS. 47A, 48 and49) or the primer (FIGS. 47B, 50 and 51) is attached to the nanopore.The advantage of the former is that capture of the tags by the nanoporeshould be fast and that there is essentially unlimited flexibility withregard to the length of the DNA to be sequenced, since the enzyme activesite and thus the tag release site will always be positioned near thenanopore channel. Thus this approach will be ideal for long sequencereads. In the latter case, the approach will be adequate for shortsequences including single-base genotyping assays. Also, in two of thefollowing embodiments (FIGS. 48 and 50), 4 tags will be attached to 4anchors which are in turn tethered to 4 distinct nucleotides; in theother two embodiments (FIGS. 49 and 51), 3 tags will be attached to 3anchors which are in turn tethered to 3 of the nucleotides, while the4^(th) nucleotide will only possess a reversible blocking group (linker)without an anchor or tag. All four sequencing schemes allow additions ofmixtures of the four nucleotides and mixtures of the four or three tags,thus reducing the number of reaction steps. In the following embodimentswashing steps are performed after each step of the procedure.

Single-Molecule SBS by a Nanopore Using 3′-O-Anchor-Cleavable LinkerNucleotides (4 Anchor 4 Tag Scheme Starting from DNA Polymerase-NanoporeConjugate) (FIG. 48)

In an embodiment of the present invention, a single polymerase moleculeis covalently attached to the nanopore at an appropriate distance toallow fast capture of the tag by the nanopore. To thispolymerase-coupled nanopore embedded in a lipid bilayer, a template DNAto be sequenced along with the appropriate primer is added followed by

1) the addition of four nucleotides comprising 3′-O-cleavable linkers(DTM) attached with anchor moieties (example set in FIG. 38). Theappropriate nucleotide analogue complementary to the nucleotide residueof the single-stranded DNA (template) which is immediately 5′ to anucleotide residue of said single-stranded DNA will be incorporated byDNA polymerase at the 3′ terminal nucleotide residue of the primer, soas to form a DNA extension product. Only a single 3′-O-anchor-cleavablelinker (DTM) nucleotide will add to the primer due to the 3′-O beingblocked by a cleavable linker and anchor moiety, preventing furtherincorporation in this step;

2) the addition to the extended primer of 4 different nanopore tagsattached with different binding molecules corresponding to 4 anchors:the appropriate binding molecule with tag will either covalently bind orcomplex with the Y-O-anchor nucleotide incorporated in step (1):

3) application of a voltage across the membrane and measuring anelectronic (ionic current) change across the nanopore resulting from thetag attached thereto generated in step (2) translocating through thenanopore, wherein the electronic change is different for each differenttype of tag, thereby identifying the nucleotide residue in thesingle-stranded template DNA, which is complementary to the incorporatedtagged nucleotide;

4) cleavage of the 3′-O-cleavable linker-attached tag by treatment withan appropriate cleaving agent, such as DTT, TCEP or THP, thus generatinga free 3′-OH ready for the next extension reaction. Iterativelyperforming steps (1)-(4) for each nucleotide residue of thesingle-stranded DNA being sequenced, wherein in each iteration of step(1) the 3′-O-cleavable anchor nucleotide is incorporated into the DNAextension product resulting from the previous iteration of step (4) ifit is complementary to the nucleotide residue of the single-stranded(template) DNA which is immediately 5′ to a nucleotide residue of saidsingle-stranded DNA hybridized to the 3′ terminal nucleotide residue ofthe DNA extension product, thereby determining the nucleotide sequenceof the single-stranded DNA.

Single-Molecule SBS by a Nanopore Using 3′-O-Anchor-Cleavable LinkerNucleotides (3 Anchor 3 Tag Scheme Starting from DNA Polymerase-NanoporeConjugate) (FIG. 49)

In another embodiment of the present invention, a single polymerasemolecule is covalently attached to the nanopore at an appropriatedistance to allow fast capture of the tag by the nanopore. To thispolymerase coupled nanopore embedded in a lipid bilayer, a template DNAto be sequenced along with the appropriate primer is added followed by

1) the addition of four nucleotides comprising one 3′-O-cleavable linker(DTM) nucleotide without anchor and three 3′-O-cleavable linker (DTM)nucleotides attached with anchor moieties via the cleavable linker(example set in FIG. 39). The appropriate nucleotide analogcomplementary to the nucleotide residue of the single-stranded(template) DNA which is immediately 5′ to a nucleotide residue of saidsingle-stranded DNA will be incorporated by DNA polymerase at the 3′terminal nucleotide residue of the primer, so as to form a DNA extensionproduct. Only a single 3′-O-anchor-cleavable linker (DTM) nucleotide orone 3′-O-cleavable linker (DTM) nucleotide (without anchor) will beadded to the primer due to the 3′-O being blocked by an anchorlesscleavable linker or a cleavable linker with anchor moiety, preventingfurther incorporation in this step:

2) addition to the extended primer of 3 different nanopore tagsconjugated with different binding molecules corresponding to the 3anchors; the appropriate binding molecule with tag will eithercovalently bind or complex with the 3′-O-anchor nucleotide incorporatedin step (1);

3) application of a voltage across the membrane and measuring anelectronic (ionic current) change across the nanopore resulting from thetag attached thereto generated in step (2) translocating through thenanopore, wherein the electronic change is different for each differenttype of tag, thereby identifying the nucleotide residue in thesingle-stranded template DNA, which is complementary to the incorporatedtagged nucleotide; if no electronic change across the nanopore can bemeasured after applying a voltage across the membrane, the incorporatednucleotide will be determined as the 3′-O-cleavable linker nucleotidewithout an anchor;

4) cleavage of the 3′-O-cleavable linker attached tag or 3′-O-cleavablelinker by treatment with appropriate cleaving agent, such as DTT, TCEPor THP, thus generating a free 3′-OH ready for the next extensionreaction.

Iteratively performing steps (1) to (4) for each nucleotide residue ofthe single-stranded DNA being sequenced, wherein in each iteration ofstep (1) the 3′-O-cleavable anchor nucleotide or 3′-O-cleavable linkernucleotide without anchor is incorporated into the DNA extension productresulting from the previous iteration of step (4) if it is complementaryto the nucleotide residue of the single-stranded (template) DNA which isimmediately 5′ to a nucleotide residue of said single-stranded DNAhybridized to the 3′ terminal nucleotide residue of the DNA extensionproduct, thereby determining the nucleotide sequence of thesingle-stranded DNA.

Single-Molecule SBS by a Nanopore Using 3′-O-Anchor-Cleavable LinkerNucleotides (4 Anchor 4 Tag Scheme Starting from DNA Primer-NanoporeConjugate) (FIG. 50)

In another embodiment of the present invention, a single stranded primercomplementary to the single stranded DNA to be sequenced is covalentlyattached to the nanopore. To this primer coupled nanopore embedded in alipid bilayer, a template DNA to be sequenced along with the DNApolymerase is added followed by

1) the addition of four nucleotides comprising 3′-O-cleavable linker(DTM) attached with anchor moieties (example set in FIG. 38). Theappropriate nucleotide analog complementary to the nucleotide residue ofthe single-stranded DNA (template) which is immediately 5′ to anucleotide residue of the single-stranded DNA will be incorporated byDNA polymerase at the 3′ terminal nucleotide residue of the primer, soas to form a DNA extension product. Only a single 3′-O-anchor-cleavablelinker (DTM) nucleotide will be added to the primer due to the 3′-Obeing blocked by a cleavable linker and anchor moiety, preventingfurther incorporation in this step:

2) the addition to the extended primer of 4 different nanopore tagsattached with different binding molecules corresponding to 4 anchors;the appropriate binding molecule with tag will either covalently bind orcomplex with the 3′-O-anchor nucleotide incorporated in step (1);

3) application of a voltage across the membrane and measuring anelectronic (ionic current) change across the nanopore resulting from thetag attached thereto generated in step (2) translocating through thenanopore, wherein the electronic change is different for each differenttype of tag, thereby identifying the nucleotide residue in thesingle-stranded template DNA, which is complementary to the incorporatedtagged nucleotide;

4) cleavage of the 3-O-cleavable linker attached tag by treatment withan appropriate cleaving agent, such as DTT, TCEP or THP, thus generatinga free 3′-OH ready for the next extension reaction.

Iteratively performing steps (1) to (4) for each nucleotide residue ofthe single-stranded DNA being sequenced, wherein in each iteration ofstep (1) the 3′-O-cleavable anchor nucleotide is incorporated into theDNA extension product resulting from the previous iteration of step (4)if it is complementary to the nucleotide residue of the single-stranded(template) DNA which is immediately 5′ to a nucleotide residue of saidsingle-stranded DNA hybridized to the 3′ terminal nucleotide residue ofthe DNA extension product, thereby determining the nucleotide sequenceof the single-stranded DNA.

Single-Molecule SBS by a Nanopore Using 3′-O-Anchor-Cleavable LinkerNucleotides (3 Anchor 3 Tag Scheme Starting from DNA Primer-NanoporeConjugate) (FIG. 51)

In another embodiment of the present invention, a single stranded primercomplementary to the single stranded DNA to be sequenced is covalentlyattached to the nanopore. To this primer coupled nanopore embedded in alipid bilayer, a template DNA to be sequenced along with the DNApolymerase is added followed by

1) the addition of four nucleotides comprising one 3′-O-cleavable linker(DTM) nucleotide without anchor and three 3′-O-cleavable linker (DTM)nucleotides attached with anchor moieties via the cleavable linker(example set in FIG. 39). The appropriate nucleotide analogcomplementary to the nucleotide residue of the single-stranded(template) DNA which is immediately 5′ to a nucleotide residue of saidsingle-stranded DNA will be incorporated by DNA polymerase at the 3′terminal nucleotide residue of the primer, so as to form a DNA extensionproduct. Only a single 3′-O-anchor-cleavable linker (DTM) nucleotide orone 3′-O-cleavable linker (DTM) nucleotide without anchor will be addedto the primer due to the 3′-O being blocked by the anchorless cleavablelinker or a cleavable linker with anchor moiety, preventing furtherincorporation;

2) addition to the extended primer of 3 different nanopore tagsconjugated with different binding molecules corresponding to the 3anchors; the appropriate binding molecule with tag will eithercovalently bind or complex with the 3′-O-anchor nucleotide incorporatedin step (1);

3) application of a voltage across the membrane and measuring anelectronic (ionic current) change across the nanopore resulting from thetag attached thereto generated in step (2) translocating through thenanopore, wherein the electronic change is different for each differenttype of tag, thereby identifying the nucleotide residue in thesingle-stranded template DNA, which is complementary to the incorporatedtagged nucleotide; if no electronic change across the nanopore can bemeasured after applying a voltage across the membrane, the incorporatednucleotide will be determined as the 3′-O-cleavable linker nucleotidewithout an anchor;

4) cleavage of the 3′-O-cleavable linker attached tag or 3′-O-cleavablelinker without tag by treatment with an appropriate cleaving agent, suchas DTT, TCEP or THP, thus generating a free 3′-OH ready for the nextextension reaction.

Iteratively performing steps (1) to (4) for each nucleotide residue ofthe single-stranded DNA being sequenced, wherein in each iteration ofstep (1) the 3-O-cleavable anchor nucleotide or 3′-O-cleavable linkernucleotide without anchor is incorporated into the DNA extension productresulting from the previous iteration of step (4) if it is complementaryto the nucleotide residue of the single-stranded (template) DNA which isimmediately 5′ to a nucleotide residue of said single-stranded DNAhybridized to the 3′ terminal nucleotide residue of the DNA extensionproduct, thereby determining the nucleotide sequence of thesingle-stranded DNA

Single-Molecule SBS by a Nanopore Using 3′-O-Anchor-Cleavable LinkerNucleotides (2 Anchor 2 Tae 2 Cleavable Linker Scheme Starting from DNAPolymerase-Nanopore Conjugate) (FIGS. 52A-52C)

In addition to the above four embodiments for four-tag and three-tagsingle-molecule SBS by a nanopore, we propose a two-tag approach (FIG.52) involving the use of two pairs of 3′-O-Anchor-Cleavable LinkerNucleotides, one pair bearing a DTM(SS) cleavable linker and the otherpair an orthogonal cleavable linker (2-nitrobenzyl (2NB) versionpresented here (FIG. 42) but other possibilities include allyl and azoversions). Another aspect of this approach is that two nucleotides bearone type of anchor molecule attached to the linker and the other twonucleotides bear a second type of anchor molecule (here we show N, andTCO anchors, but other possible anchors comprise biotin, PBA,quadricyclane and norbornene). Finally two different tags are used thatproduce different nanopore electronic (ionic current) signals (here weshow TAG1 attached to DBCO and TAG2 attached to Tetrazine, but otherpossible anchor binding molecules comprise streptavidin, SHA,bis(dithiobenzil)nickel(II) compounds and nitrile oxide). The DBCO groupon TAG1 conjugates with the N₃ anchor and the Tetrazine group on TAG2conjugates with the TCO anchor. Importantly, each nucleotide will have aunique combination of cleavable linker and anchor: in the examplepresented, we use a N₃ anchor and SS cleavable linker on dATP, a TCOanchor and SS cleavable linker on dCTP, a N₃ anchor and 2NB cleavablelinker on dGTP, and a TCO anchor and 2NB cleavable linker on dTTP.Finally, while the following embodiment is illustrated with a polymerasemolecule covalently attached to the nanopore as in FIG. 47a , it canalso be performed with the primer attached to the nanopore as in FIG.47b . In the following embodiment washing steps are performed after eachstep of the procedure.

In an embodiment of the present invention, a single polymerase moleculeis covalently attached to the nanopore at an appropriate distance toallow fast capture of the tag by the nanopore. To thispolymerase-coupled nanopore embedded in a lipid bilayer, a template DNAto be sequenced along with the appropriate primer is added followed by

1) the addition of four nucleotides comprising cleavable linkers andanchor moieties as follows (3′-O—N₃-SS-dATP, 3′-O-TCO-SS-dCTP,3′-O—N₃-2NB-dGTP and 3′-O-TCO-2NB-dTTP) (example set in FIG. 42). Theappropriate nucleotide analogue complementary to the nucleotide residueof the single-stranded DNA (template) which is immediately 5′ to anucleotide residue of said single-stranded DNA will be incorporated byDNA polymerase at the 3′ terminal nucleotide residue of the primer, soas to form a DNA extension product. Only a single 3′-O-anchor-cleavablelinker nucleotide will add to the primer due to the 3′-O being blockedby a cleavable linker and anchor moiety, preventing furtherincorporation in this step:

2) the addition to the extended primer of 2 different nanopore tagsattached with different binding molecules corresponding to 2 anchors(DBCO-TAG1 and Tetrazine-TAG2); the appropriate binding molecule withtag will either covalently bind or complex with the 3′-O-anchornucleotide incorporated in step (1); thus, A and G will receive TAG1, Cand T will receive TAG2.

3) application of a voltage across the membrane and measuring anelectronic (ionic current) change across the nanopore resulting from thetag attached thereto generated in step (2) translocating through thenanopore, wherein the electronic change is different for the twodifferent tags, thereby partially identifying the nucleotide residue inthe single-stranded template DNA, which is complementary to theincorporated tagged nucleotide; thus a TAG1 signal will indicate thateither A or G was incorporated, a TAG2 signal will indicate that eitherC or T was incorporated.

4) cleavage of the 3′-O-2-nitrobenzyl linker-attached tags by treatmentwith light at ˜340 nm, thus generating a free 3′-OH ready for the nextextension reaction on primers that were extended with G or T, whileleaving tags on A and C.

5) application of a voltage across the membrane and measuring anelectronic (ionic current) change across the nanopore resulting from anytag still attached thereto generated in step (2) translocating throughthe nanopore, wherein the electronic change is different for the twodifferent tags, thereby identifying the nucleotide residue in thesingle-stranded template DNA, which is complementary to the incorporatedtagged nucleotide; thus loss of a TAG1 signal seen in step (3) willindicate that a G was incorporated while a remaining TAG1 signal willindicate that an A was incorporated; loss of a TAG2 signal seen in step(3) will indicate that T was incorporated while a remaining TAG2 signalwill indicate that C was incorporated.

6) cleavage of the 3′-O-SS(DTM) linkers from any remaining tags bytreatment with DTT or TCEP or THP to restore the 3′-OH group inreadiness for the next cycle of SBS sequencing.

Iteratively performing steps (1)-(6) for each nucleotide residue of thesingle-stranded DNA being sequenced, wherein in each iteration of step(1) the 3′-O-cleavable anchor nucleotide is incorporated into the DNAextension product resulting from the previous iteration of step (4) ifit is complementary to the nucleotide residue of the single-stranded(template) DNA which is immediately 5′ to a nucleotide residue of saidsingle-stranded DNA hybridized to the 3′ terminal nucleotide residue ofthe DNA extension product, thereby determining the nucleotide sequenceof the single-stranded DNA.

REFERENCES FOR EXAMPLE 3 INCLUDE

Kumar et al. PEG-labeled nucleotides and nanopore detection for singlemolecule DNA sequencing by synthesis. Scientific Reports (2012) 2, 684.Fuller et al. Real-time single-molecule electronic DNA sequencing bysynthesis using polymer-tagged nucleotides on a nanopore array.Proceedings of the National Academy of Sciences U.S.A. (2016) 113,5233-5238. Fuller et al. Chemical methods for producing taggednucleotides. US Patent Application US20150368710. Zheng et al.Development of bioorthogonal reactions and their applications inbioconjugation. Molecules (2015) 20, 3190-3205. Springer et al.Salicylhydroxamic acid functionalized affinity membranes for specificimmobilization of proteins and oligonucleotides. Journal of BiomolecularTechniques (2003) 14, 183-190. Sletten EM, Bertozzi CR. A bioorthogonalquadricyclane ligation. Journal of the American Chemical Society (2011)133, 17570-17573. Gutsmiedl K et al. Copper-free “click” modification ofDNA via nitrile oxide-norbornene 1,3-dipolar cycloaddition. OrganicLetters (2009) 11, 2405-2408.

Example 4: Fluorescence-Based SBS Sequencing with 3′-O-Dye-SS(DTM)-dNTPsand 3′-O-Anchor-SS(DTM)-dNTPs

We present results for two of the above described schemes involvingpairs of 3′-O-Dye-SS(DTM)-dNTPs for two of the four bases (for example,A and T (or U)) and 3′-O-Anchor-SS(DTM)-dNTPs for the other two bases(for example, C and G). First, we demonstrate the ability to incorporateall four of these in succession using solution-based assays and aMALDI-TOF MS readout (FIG. 53). Next we present the ability to obtain a4-base read (FIG. 54) for a four-color SBS protocol (FIG. 70) withtemplates immobilized on glass slides and fluorescence scanning usingfour dyes that have distinct emission spectra. Then we demonstrate 4 and6-base reads (FIG. 56) obtained using a set of dyes that generates onlytwo color signals (FIG. 71).

We also propose seven new schemes in this document taking advantage ofthe use of the following types of nucleotide analogues in variouscombinations: (1) those with dyes attached to the 5 position ofpyrimidine bases or 7 position of purine bases via azo linkages andhaving dithiomethyl blocking groups with attached dyes at the3′-O-position (3′-O-DTM-dNTP-SS-Dyes and 3′-O-DTM-dNTP-Azo-Dyes); (2)those with dyes directly attached to the 3′-O-group on the sugar viadithiomethyl-based linkers (3′-O-Dye-SS(DTM)-dNTPs); (3) those withanchors for subsequent attachment of dyes attached to the 3′-O-positionvia dithiomethyl groups, allyl, or 2-nitrobenzyl groups(3′-O-Anchor-SS(DTM)-dNTPs, 3′-O-Anchor-Allyl-dNTPs,3′-O-Anchor-2-Nitrobenzyl-dNTPs). Both 4-color and 2-color variants ofSBS sequencing reactions are described using sets of these nucleotides:For four color versions, the use of either two from group (1) and twofrom group (2) (FIG. 72) or two from group (1) and two from group (3)(FIG. 73) are described. For two color versions, the use of two fromgroup (1) and two from group (3) (FIG. 74), two from group (1) and twofrom group (2) (FIG. 75), or four from group (3), two of which have SSand two of which have 2NB (or other) linkages (FIG. 76) are given asexamples.

Demonstration of Incorporation of Two 3′-O-Dye-SS(DTM)-dNTPs and Two3′-O-Anchor-SS(DTM)-dNTPs to Obtain a Continuous Four Base Sequence inSolution:

FIG. 53. Use of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-PEG₄-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dGTP and3′-O-Biotin-SS-dCTP) for continuous SBS with MALDI-TOF MS detection ofintermediate products. Reactions were carried out in solution withmixtures of two 3′-dye modified nucleotides (3′-SS-Rox-dATP and3′-SS-BodipyFL-dTTP) and two 3′-anchor modified nucleotides(3′-SS-Biotin-dCTP and 3′-SS-TCO-dGTP). Replicate reactions consisted of20 pmol of the 51mer template shown below, 100 pmol primer orbase-extended primers (13-16mer), 150 pmol 3′-O-Dye(Anchor)-dNTPsmixture, 2 units Therminator IX DNA polymerase and 2 mM manganese in 20μl 1× Thermo Pol buffer subjected to 38 cycles of 30 sec at 65° C. and30 sec at 45° C. Reactions from multiple replicate tubes were pooled andHPLC was used to remove unused 3′-Dye(Anchor)-dNTPs and salt and obtainpure incorporation products as verified by MALDI-TOF MS. Cleavage with100 pmol tris-hydroxypropyl phosphine (THP) for 5 min at 65° C. led torecovery of the 3′ OH. The samples were treated with OligoClean &Concentrator™ kit (ZymoResearch, USA) to remove salt and cleaved groupsand sizes of products checked by MALDI-TOF MS. The 13-mer shown belowwas used in the initial reaction. In subsequent cycles, primers extendedat the 3′ end with the base from the previous cycle were used.

As shown in the scheme at the left, 4 cycles of extension (a, c, e, g)and cleavage (b, d, f, g) were conducted to add A, C, G and T to the 3′ends of these primers (complementary to the 4 bases 5′ to the underlinedprimer binding site shown in bold letters in the template). The resultsof MALDI-TOF MS analysis confirmed that the correct nucleotides wereadded and then converted to natural nucleotides containing a free 3′-OHgroup in each cycle. Addition of the nucleotide mixture to the 13-merprimer annealed to a DNA template resulted in complete incorporation of3′-SS-PEG4-Rox-dATP into the primer as evidenced by the single observedpeak in the mass spectrum (MS) of 5188 Da (5188 Da expected) (a). Aftertreatment with THP to cleave the 3′-SS-PEG4-Rox group, a single MS peakwas observed at 4264 Da (4272 Da expected) (b). Extension of the 14-merprimer in the second cycle revealed incorporation of 3′-SS-Biotin-dCTPinto the growing primer strand (single MS peak at 4941 Da observed, 4939Da expected) (c). After treatment with THP, a single cleavage peak at4564 Da was found (4561 Da expected) (d). In the third cycle,incorporation of 3′-SS-TCO-dGTP generated a MS peak of 5184 Da (5194 Daexpected) (e) and complete cleavage of the anchor and restoration of the3′-OH group (MS peak at 4894 Da, 4890 Da expected) was shown by MS (f).Finally, in the fourth cycle, the newly formed 16-mer DNA strand wasused as a primer for 3′-SS-BodipyFL-dTTP incorporation. The MS results(g and h) demonstrated a single peak with molecular weight of 5621 Da(5620 Da expected) for 3′-SS-BodipyFL-dTTP incorporation and 5197 Da(5195 Da expected) after cleavage.

51mer template: 5′-TACATCAACTACCCGGAGGCCAAGTACGGCGGGTACGT CCTTGACAATGTG-3′ 13mer primer: 5′-CACATTGTCAAGG-3′ MW: 3959After each incorporation, the expected size of the product should be thesum of the starting primer plus the incoming nucleotide minus the MW(175) of the pyrophosphate group, yielding MWs of 5188 Da, 4939 Da, 5194Da and 5620 Da.

Demonstration of 4-Color Sequencing Using a Combination of3′-O-Dye-SS(DTM)-dNTPs and 3′-O-Anchor-SS(DTM)-dNTPs with theirCorresponding Dye Labeled Binding Molecules on a DNA Primer-LoopTemplate Immobilized on Glass Slides (FIG. 70 and FIG. 54)

Scheme 1.

Use of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-PEG₄-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dGTP and3′-O-Biotin-SS-dCTP) with their corresponding Dye Labeled BindingMolecules (TAMRA Labeled Tetrazine and Cy5 Labeled Streptavidin) toperform 4-color DNA SBS. Step 1, addition of DNA polymerase and the fournucleotide analogues (3′-O-Rox-PEG₄-SS-dATP, 3′-O-BodipyFL-SS-dTTP,3′-O-TCO-SS-dGTP and 3′-O-Biotin-SS-dCTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand to terminate DNA synthesis. Step 2,Chase: addition of the DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS(DTM)-nucleotide analogue to the growing DNAstrands that were not extended with one of the dye or anchor labeleddNTPs in step 1. The growing DNA strands are terminated with one of thefour dye or anchor labeled nucleotide analogues (A, C, G, T) or the sameone of the four nucleotide analogues (A, C, G, T) without dye or anchor.Step 3, Next, the dye labeled binding molecules (TAMRA labeled tetrazineand Cy5 labeled streptavidin) are added to the DNA extension products,which will specifically connect with the two unique “anchor” moieties(TCO and biotin) on each DNA extension product, to enable the labelingof each DNA product terminated with each of the two nucleotide analogues(G and C) with two distinct fluorescent dyes (labeled with TAMRA for Gand labeled with Cy5 for C). Step 4, after washing away the unbound dyelabeled binding molecules, detection of the unique fluorescence signalfrom each of the fluorescent dyes on the DNA products allows theidentification of the incorporated nucleotide for sequencedetermination. Next, in Step 5, treatment of the DNA products with THPcleaves the SS linker, leading to the removal of the fluorescent dye andthe regeneration of a free 3′-OH group on the DNA extension product,which is ready for the next cycle of the DNA sequencing reaction.Structures of modified nucleotides used in this scheme are shown in FIG.55.

FIG. 56: Four base read for SBS of DNA immobilized on slides usingfour-color approach. Using the primer-loop template shown at the top ofthe figure, in which the next four bases to be added are C, A, T, C,reactions were carried out as in the protocol for FIG. 70.5′-NH₂-modified template was immobilized on NHS ester-modified slidesfrom Surmodics. Each cycle was carried out as follows: (1) extensionwith 60 μl of 0.02 μM 3′-O-Rox-PEG₄-SS-dATP, 0.05 μM3′-O-BodipyFL-SS-dTTP, 0.5 μM 3′-O-Biotin-PEG₄-SS-dCTP, 0.5 μM3′-O-TCO-SS-dGTP, 1× Thermo Pol Reaction Buffer (NEB), 2 mM MnCl₂, 6 UTherminator IX DNA polymerase for 15 min at 65° C.; (2) washing with 1×Thermo Pol Reaction Buffer; (3) chase with 60 μl of 4 μM each of thefour 3′-O-SS(DTM)-dNTPs, 1× Thermo Pol Reaction Buffer, 2 mM MnCl₂, 6 UTherminator IX DNA polymerase for 10 min at 65° C.; (4) washing with 1×Thermo Pol Reaction Buffer; (5) labeling with 60 μl of 10 μMTetrazine-PEG₄-TAMRA, 4 μM Streptavidin-Cy5, 1×PBS, pH 7.4 for 10 min at37° C.; (6) washing with 1× Thermo Pol Reaction Buffer, 1×SPSC buffer(1×PBS, pH 7.4, 0.5M NaCl, 0.1% Tween-20) and water; (7) scanning airdried slides with excitation at 488 nm, 543 nm, 594 nm and 633 nm, andemission settings at appropriate wavelengths for each dye, to recordfluorescence intensity of spots; (8) cleavage with 10 mM THP for 10 minat 65° C.; (9) washing with water, 1×SPSC, and water again; (9) scanningair dried slides to determine background (repeating washes as necessaryto minimize the background). The above was carried out 4 times to obtainthe raw image intensity readings shown in the bar graph at the bottomfor the first four bases of the extended primer.

FIG. 55: Structures of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-PEG4-SS-dGTPand 3′-O-Biotin-SS-dCTP) with their corresponding Dye Labeled BindingMolecules (TAMRA or Cy3 Labeled Tetrazine and Cy5 Labeled Streptavidin)to perform 4-color DNA SBS using approach delineated in FIG. 70.

Demonstration of Successful 2-Color Continuous Sequencing Using aCombination of 3′-O-Dye-SS(DTM)-dNTPs and 3′-O-Anchor-SS(DTM)-dNTPs withtheir Corresponding Dye Labeled Binding Molecules on Immobilized DNATemplates (FIG. 71 and FIG. 56):

FIG. 71. Use of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-PEG₄-SS-dGTPand 3′-O-Biotin-PEG₄-SS-dCTP) with their corresponding Dye LabeledBinding Molecules (Alexa488-PEG₄ Labeled Tetrazine and Alexa594 LabeledStreptavidin) to perform 2-color DNA SBS. Although 4 different dyes havebeen used in this experiment, Rox and Alexa594 have very similarabsorption and emission spectra, as do BodipyFL and Alexa488. Hence thisis described as a 2-color experiment. Step 1, addition of DNA polymeraseand the four nucleotide analogues (3′-O-Rox-SS-dATP,3′-O-BodipyFL-SS-dTTP, 3′-O-TCO-PEG₄-SS-dGTP and3′-O-Biotin-PEG₄-SS-dCTP) to the immobilized primed DNA template enablesthe incorporation of the complementary nucleotide analogue to thegrowing DNA strand to terminate DNA synthesis. Step 2. Chase: additionof the DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the growing DNA strandsthat were not extended with one of the dye or anchor labeled dNTPs instep 1. The growing DNA strands are terminated with one of the four dyeor anchor labeled nucleotide analogues (A, C, G, T) or the same one ofthe four nucleotide analogues (A, C, G, T) without dye or anchor.Imaging is performed to identify incorporation of 3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP. Step 3, Next, the dye labeled binding molecules(Alexa488-PEG₄ labeled tetrazine and Alexa594 labeled streptavidin) areadded to the DNA extension products, which will specifically connectwith the two unique “anchor” moieties (TCO and biotin) on each DNAextension product, to enable the labeling of each DNA product terminatedwith each of the two nucleotide analogues (G and C) with two distinctfluorescent dyes (labeled with Alexa488 for G and labeled with Alexa594for C). Step 4, after washing away the unbound dye labeled bindingmolecules, detection of the unique fluorescence signal from each of thefluorescent dyes on the DNA products allows the identification of theincorporated nucleotide for sequence determination. Next, in Step 5,treatment of the DNA products with THP cleaves the SS linker, leading tothe removal of the fluorescent dye and the regeneration of a free 3′-OHgroup on the DNA extension product, which is ready for the next cycle ofthe DNA sequencing reaction. Structures of modified nucleotides used inthis scheme are shown in FIG. 71.

FIG. 56: Four and six base reads for SBS of primer-loop template DNAimmobilized on slides using two-color approach. Using the looped primingtemplate shown at the top of the figure, in which the next four bases tobe added are T, A, G, A, or the looped priming template shown in themiddle of the figure, in which the next six bases are C, A, T, C, A, A,reactions were carried out as in the protocol for FIG. 71.5′-NH₂-modified template was immobilized on NHS ester-modified slidesfrom Surmodics. Each cycle was carried out as follows: (1) extensionwith 60 μl of 0.02 μM 3′-O-Rox-PEG₄-SS-dATP, 0.05 μM3′-O-BodipyFL-SS-dTTP, 0.5 M 3′-O-Biotin-SS-dCTP, 0.2 μM3′-O-TCO-SS-dGTP, 1× Thermo Pol Reaction Buffer (NEB), 2 mM MnCl₂, 6 UTherminator IX DNA polymerase for 15 min at 65° C.; (2) washing with IXThermo Pol Reaction Buffer; (3) chase with 60 μl of 4 μM each of thefour 3′-O-SS(DTM)-dNTPs, 1× Thermo Pol Reaction Buffer, 2 mM MnCl₂, 6 UTherminator IX DNA polymerase for 10 min at 65° C.; (4) washing with 1×Thermo Pol Reaction Buffer; (5) scanning air dried slides at 488 nm and594 nm excitation with appropriate emission settings to recordfluorescence intensity of spots; (6) labeling with 60 μl of 0.5 μMTetrazine-PEG4-Alexa488, 0.5 μM Streptavidin-Alexa594, 1×PBS, pH 7.4 for10 min at 37° C.; (7) washing with 1× Thermo Pol Reaction Buffer, 1×SPSCbuffer and water; (8) scanning air dried slides at 488 nm and 594 nmexcitation with appropriate emission settings to record fluorescenceintensity of spots; (9) cleavage with 5 mM THP for 10 min at 65° C.;(10) washing with water, 1×SPSC, and water again; (11) scanning airdried slides to determine background (repeating washes as necessary toobtain minimal background). The above was carried out 4-6 times toobtain the raw image intensity readings shown in the bar graphs belowthe template structures. In each cycle, E represents the imaging resultsafter the extension and L represents the imaging results after thelabeling. So in the top graph, the T is determined after the initialextension due to the presence of the BodipyFL dye directly attached tothe 3′-O- of the dTTP, as are the A's in the second and fourth cycle;however the G in the third cycle is not seen until the labeling reactionin which the Alexa488-tetrazine is conjugated to the anchoring molecule(TCO) on the 3′-O- of the dGTP. Similarly in the lower bar graph, theA's and T's are visualized immediately after extension, but the C's arenot observed until the labeling reaction is performed.

FIG. 57: Structures of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP), 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-PEG₄-SS-dGTPand 3′-O-Biotin-SS-dCTP) with their corresponding Dye Labeled BindingMolecules (Alexa488 Labeled Tetrazine and Alexa594 Labeled Streptavidin)to perform 2-color DNA SBS using approach delineated in FIG. 71.

FIG. 72: Use of 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5,3′-O-SS-dCTP-5-SS-R6G); 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-PEG₄-SS-dATPand 3′-O-BodipyFL-SS-dTTP) for 4-color DNA SBS. Step 1, Addition of DNApolymerase and the four nucleotide analogues (3′-O-SS-dGTP-7-SS-Cy5,3′-O-SS-dCTP-5-SS-R6G, 3′-O-Rox-PEG₄-SS-dATP and 3′-O-BodipyFL-SS-dTTP)to the immobilized primed DNA template enables the incorporation of thecomplementary dye labeled nucleotide analogue to the growing DNA strand.The growing DNA strand is terminated with one of the four nucleotideanalogues (A, C, G, T) with the four distinct fluorescent dyes. Step 2,Chase: addition of DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the subset of growing DNAstrands in the ensemble that were not extended with any of the dyelabeled dNTPs in step 1. The growing DNA strands are terminated with oneof the four nucleotide analogues (A, C, G, T) with the four distinctfluorescent dyes or the same one of the four nucleotide analogues (A, C,G, T) without dye. Step 3, after washing away the unincorporatednucleotide analogues, detection of the unique fluorescence signal fromeach of the fluorescent dyes on the DNA products allows theidentification of the incorporated nucleotide for sequencedetermination. Next, in Step 4, treatment of the DNA products with THPcleaves the SS linker, leading to the removal of the fluorescent dye andthe regeneration of a free 3′-OH group on the DNA extension product,which is ready for the next cycle of the DNA sequencing reaction.Structures of modified nucleotides used in this scheme are shown in FIG.58.

FIG. 58: Structures of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP) and 3′-O-SS(DTM)-dNTP-SS-Dyes(3′-O-SS-dGTP-7-SS-Cy5 and 3′-O-SS-dCTP-5-SS-R6G) for 4-color sequencingusing approach delineated in FIG. 72.

FIG. 73: Use of 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5,3′-O-SS-dCTP-5-SS-R6G); 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP,3′-O-Biotin-SS-dTTP) with their corresponding Dye Labeled BindingMolecules (Rox Labeled Tetrazine and BodipyFL Labeled Streptavidin) toperform 4-color DNA SBS. Step 1, addition of DNA polymerase and the fournucleotide analogues (3′-O-SS-dGTP-7-SS-Cy5, 3′-O-SS-dCTP-5-SS-R6G,3′-O-TCO-SS-dATP and 3′-O-Biotin-SS-dTTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand to terminate DNA synthesis. Step 2.Chase: addition of DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS(DTM)-nucleotide analogue to the subset of growingDNA strands in the ensemble that were not extended with any of the dyelabeled dNTPs in step 1. The growing DNA strands are terminated with oneof the four dye or anchor labeled nucleotide analogues (A, C, G, T) orthe same one of the four nucleotide analogues (A, C, G, T) without dyeor anchor. Step 3, next, the dye labeled binding molecules (Rox labeledtetrazine and BodipyFL labeled streptavidin) are added to the DNAextension products, which will specifically connect with the two unique“anchor” moieties (TCO and biotin) on each DNA extension product, toenable the labeling of each DNA product terminated with each of the twonucleotide analogues (A and T) with two distinct fluorescent dyes(labeled with Rox for A and labeled with BodipyFL for T). Step 4, afterwashing away the unbound dye-labeled binding molecules, detection of theunique fluorescence signal from each of the fluorescent dyes on the DNAproducts allows identification of the incorporated nucleotides forsequence determination. A Rox signal indicates incorporation of A, aBodipyFL signal indicates incorporation of T, a Cy5 signal indicatesincorporation of G and an R6G signal indicates incorporation of C. Next,in Step 5, treatment of the DNA products with THP cleaves the SS linker,leading to the removal of the remaining fluorescent dye and theregeneration of a free 3′-OH group on the DNA extension product, whichis ready for the next cycle of the DNA sequencing reaction. Structuresof modified nucleotides used in this scheme are shown in FIG. 59.

FIG. 59: Structures of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP and3′-O-Biotin-SS-dTTP), 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5and 3′-O-SS-dCTP-5-SS-R6G) and the corresponding Dye Labeled BindingMolecules (Rox Labeled Tetrazine and BodipyFL Labeled Streptavidin) toperform 4-color DNA SBS using approach delineated in FIG. 73.

FIG. 74: Use of 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5,3′-O-SS-dCTP-5-SS-R6G); 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP,3′-O-Biotin-SS-dTTP) with their corresponding Dye Labeled BindingMolecules (Cy5 Labeled Tetrazine and R6G Labeled Streptavidin) toperform 2-color DNA SBS. Step 1, addition of DNA polymerase and the fournucleotide analogues (3′-O-SS-dGTP-7-SS-Cy5, 3′-O-SS-dCTP-5-SS-R6G,3′-O-TCO-SS-dATP and 3′-O-Biotin-SS-dTTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand to terminate DNA synthesis. Step 2,Chase: addition of DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the subset of growing DNAstrands in the ensemble that were not extended with any of the dye oranchor labeled dNTPs in step 1. The growing DNA strands are terminatedwith one of the four dye labeled nucleotide analogues (A, C, G, T) orthe same one of the four nucleotide analogues (A, C, G, T) without dye.Step 3, after washing away the unincorporated dye labeled nucleotides,detection of the unique fluorescence signal from each of the fluorescentdyes on the DNA products allows identification of the incorporatednucleotide for sequence determination, Cy5 signal indicatesincorporation of G, R6G signal indicates incorporation of C. Step 4,next, the dye labeled binding molecules (Cy5 labeled tetrazine and R6Glabeled streptavidin) are added to the DNA extension products, whichwill specifically connect with the two unique “anchor” moieties (TCO andbiotin) on each DNA extension product, to enable the labeling of eachDNA products terminated with one of the two nucleotide analogues (A andT) with two distinct fluorescent dyes (labeled with Cy5 for A andlabeled with R6G for T). Step 5, after washing away the unattachedlabels, a second round of detection of the unique fluorescence signalfrom each of the fluorescent dyes on the DNA products allows theidentification of the incorporated nucleotide for sequencedetermination. Appearance of a Cy5 signal indicates incorporation of A,R6G signal indicates incorporation of T. Next, in Step 6, treatment ofthe DNA products with THP cleaves the SS linker, leading to the removalof the remaining fluorescent dye and the regeneration of a free 3′-OHgroup on the DNA extension product, which is ready for the next cycle ofthe DNA sequencing reaction. Structures of modified nucleotides used inthis scheme are shown in FIG. 60.

FIG. 60: Structures of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O-TCO-SS-dATP and3′-O-Biotin-SS-dTTP), 3′-O-SS(DTM)-dNTP-SS-Dyes (3′-O-SS-dGTP-7-SS-Cy5and 3′-O-SS-dCTP-5-SS-R6G) and the corresponding Dye Labeled BindingMolecules (Cy5 Labeled Tetrazine and R6G Labeled Streptavidin) toperform 2-color DNA SBS using approach delineated in FIG. 74.

FIG. 75: Use of 3′-O-SS(DTM)-dNTP-Azo-Dyes (3′-O-SS-dGTP-7-Azo-Rox,3′-O-SS-dCTP-5-Azo-BodipyFL); 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP,3′-O-BodipyFL-SS-dTTP) to perform 2-color DNA SBS. Step 1, addition ofDNA polymerase and the four nucleotide analogues(3′-O-SS-dGTP-7-Azo-Rox, 3′-O-SS-dCTP-5-Azo-BodipyFL, 3′-O-Rox-SS-dATPand 3′-O-BodipyFL-SS-dTTP) to the immobilized primed DNA templateenables the incorporation of the complementary nucleotide analogue tothe growing DNA strand to terminate DNA synthesis. Step 2. Chase:addition of DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS-nucleotide analogue to the subset of growing DNAstrands in the ensemble that were not extended with any of the dyelabeled dNTPs in step 1. The growing DNA strands are terminated with oneof the four dye labeled nucleotide analogues (A, C, G. T) or the sameone of the four nucleotide analogues (A, C, G. T) without dye. Step 3,after washing away the unincorporated dye labeled nucleotides, detectionof the unique fluorescence signal from each of the fluorescent dyes onthe DNA products allows the identification of the incorporatednucleotide for sequence determination. Rox signal indicatesincorporation of A or G, BodipyFL signal indicates incorporation of C orT. Step 4, cleavage of Azo linker by adding sodium dithionite (Na₂S₂O₄)to the elongated DNA strands results in removal of Rox from incorporatedG and BodipyFL from incorporated C. Step 5, after washing away thecleaved dyes, a second round of detection of the unique fluorescencesignal from each of the fluorescent dyes on the DNA products allows theidentification of the incorporated nucleotide for sequencedetermination. Disappearance of Rox signal indicates incorporation of G,and disappearance of BodipyFL signal indicates incorporation of C.Remaining Rox signal indicates incorporation of A, and remainingBodipyFL signal indicates incorporation of T. Next, in Step 6, treatmentof the DNA products with THP cleaves the SS linker, leading to theremoval of the remaining fluorescent dye and the regeneration of a free3′-OH group on the DNA extension product, which is ready for the nextcycle of the DNA sequencing reaction. The presence of an additional SSlinkage between the Azo group and the base results in the production ofa shorter scar on the incorporated nucleotide after THP treatment whichshould result in longer reads. Structures of modified nucleotides usedin this scheme are shown in FIG. 61.

FIG. 61: Structures of 3′-O-Dye-SS(DTM)-dNTPs (3′-O-Rox-SS-dATP and3′-O-BodipyFL-SS-dTTP) and 3′-O-DTM(SS)-dNTP-Azo-Dyes(3′-O-SS-dGTP-7-Azo-Rox or 3′-O-SS-dGTP-7-SS-Azo-Rox and3′-O-SS-dCTP-5-Azo-BodipyFL or 3′-O-SS-dCTP-5-SS-Azo-BodipyFL) for2-color DNA SBS using approach delineated in FIG. 75.

Two Color SBS Scheme Involving Two Different Anchors and Two DifferentCleavable Linkers for Dye Attachment on the 3′-O-Position of theDeoxynucleotide:

A number of different cleavable linkers can be used in this scenario.Two of the nucleotides will bear an SS(DTM) linker and the other twowill include a 2-nitrobenzyl (2NB) linker (or azo linker or allyllinker). The two linkers will be used in cross combination with twoanchor-binding molecule pairs (TCO anchor on the 3′-OH of the nucleotidewith its binding partner Tetrazine attached to one fluorescent dye; N₃anchor with its partner binding molecule attached to a second dye. Thekey is that each of the four nucleotides will have a different anchorand linker combination; for instance dATP could have N₃ anchor and SSlinker; dCTP could have TCO anchor and SS linker; dGTP could have N₃anchor and 2NB linker; dTTP could have TCO anchor and 2NB linker. Thoughwe use SS (cleaved with DTT, TCEP or THP) and 2-nitrobenzyl linkers(cleaved with ˜340 nm light) in the following scheme (FIG. 76), otherpossible choices are SS and azo linkers (cleaved with sodium dithionite)or SS and allyl linkers (cleaved with Pd(0)).

FIG. 76: Use of 3′-O-Anchor-SS(DTM)-dNTPs (3′-O—N₃-SS-dATP and3′-O-TCO-SS-dCTP) and 3′-O-Anchor-2-Nitrobenzyl-dNTPs(3′-O-N₃-2-Nitrobenzyl-dGTP and 3′-O-TCO-2-Nitrobenzyl-dTTP) with theircorresponding Dye Labeled Binding Molecules (BodipyFL Labeled DBCO andRox labeled Tetrazine) to perform 2-color DNA SBS. Step 1, addition ofDNA polymerase and the four nucleotide analogues (3′-O—N₃-SS-dATP,3′-O-TCO-SS-dCTP, 3′-O—N₃-2-Nitrobenzyl-dGTP and3′-O-TCO-2-Nitrobenzyl-dTTP) to the immobilized primed DNA templateenables the incorporation of the complementary nucleotide analogue tothe growing DNA strand to terminate DNA synthesis. Step 2, Chase:addition of DNA polymerase and four 3′-O-SS(DTM)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O-SS(DTM)-nucleotide analogue to the subset of growingDNA strands in the ensemble that were not extended with any of theanchor labeled dNTPs in step 1. The growing DNA strands are terminatedwith one of the four anchor labeled nucleotide analogues (A, C, G, T) orthe same one of the four nucleotide analogues (A, C, G, T) without dyeor anchor. Step 3, next, the dye labeled binding molecules (Rox labeledTetrazine and BodipyFL labeled DBCO) are added to the DNA extensionproducts, which will specifically connect with the two unique “anchor”moieties (TCO and N₃) on each DNA extension product, to enable thelabeling of each DNA product terminated with one of the four nucleotideanalogues with one of the two dyes (A and G with BodipyFL and C and Twith Rox). Step 4, after washing away the unbound dye-labeled bindingmolecules, detection of the fluorescence signals from each of thefluorescent dyes on the DNA products allows partial identification ofthe incorporated nucleotides for sequence determination. A BodipyFLsignal indicates incorporation of A or G, a Rox signal indicatesincorporation of T or C. Next, in Step 5, treatment of the DNA productswith 340 nm light cleaves the 2-Nitrobenzyl linker, leading to theremoval of the fluorescent dye and the regeneration of a free 3′-OHgroup on the DNA extension products extended with either a G or T. Afterwashing, in Step 6 imaging is carried out a second time to detectremaining fluorescent signals. Loss of a BodipyFL signal indicates thatthe incorporated nucleotide was a G, a remaining Bodipy FL signalindicates that the incorporated nucleotide was an A; similarly loss of aRox signal indicates that the incorporated nucleotide was a T, aremaining Rox signal indicates that the incorporated nucleotide was a C.Finally, in Step 7, treatment with THP cleaves any dye remaining onincorporated A or C, and restores the 3′-OH on those nucleotides aswell. At this point, the extension products are ready for the next cycleof the DNA sequencing reaction. Structures of modified nucleotides usedin this scheme are shown in FIG. 63.

FIG. 63: 3′-O-Anchor-SS(DTM)-dNTP (3′-O-TCO-SS-dCTP and3′-O—N₃-SS-dATP), 3′-O-Anchor-2NB-dNTPs (3′-O-TCO-2NB-dTTP and3′-O—N₃₋₂NB-dGTP) and their corresponding Dye-labeled binding molecules(Rox labeled tetrazine and BodipyFl labeled DBCO) for 2-color DNA SBSusing approach delineated in FIG. 76.

FIG. 64: Structures of 3′-O-Anchor-SS(DTM)-dNTP,3′-O-Anchor-Allyl-dNTPs, and 3′-O-Anchor-2NB-dNTPs. Combinatorial use oftwo from one category with the same anchor, two from another categorywith another anchor and their corresponding two Dye-labeled bindingmolecules results in 2-color DNA SBS. One specific approach is shown inFIG. 76 as an example.

FIG. 66: Example synthesis of 3′-O-SS(DTM)-dGTP-SS-Azo-Rox and3′-O-SS(DTM)-dTTP-SS-Azo-BodipyFL. Rox and BodipyFL labeled Azo LinkerNHS esters are coupled with 3′-O-SS(DTM)-dGTP-SS-NH₂ and3′-O-SS(DTM)-dTTP-SS-NH₂ giving 3′-O-SS(DTM)-dGTP-SS-Azo-Rox and3′-O-SS(DTM)-dTTP-SS-Azo-BodipyFL.

FIG. 67: Synthesis of 3′-O-SS(DTM)-dATP-SS-Rox.

FIG. 68: Synthesis of 3′-O-SS(DTM)-dUTP-SS-BodipyFL.

FIG. 69: Example syntheses of 3′-O-Anchor-2NB-dNTP(3′-O-TCO-2-Nitrobenzyl-dTTP and 3′-O-Azido-2-Nitrobenzyl-dGTP).

Synthetic Schemes

Additional Examples with Nucleotide Analogues that have Modifications onBoth 3′ Position and Base

Here are provided 8 additional SBS schemes using nucleotide analoguesthat include 3′ modifications (dyes or anchors) along with basemodifications (anchors or directly attached dyes). Included are twoexamples aimed mainly at single molecule sequencing in which clusters ofdyes are placed at these positions (via anchors at 3′ position ordirectly on the base). The dye clusters include multiple dyes placed invarious positions on linear polymers as well as branched polymers(dendrimers).

In the schemes described, even if the 3′ position of the nucleotideanalogues is not further modified, it will be blocked by a dithiomethyl(DTM) moiety which can be cleaved specifically with THP. In thefollowing schemes Azo, Allyl and 2-nitrobenzyl groups are used asexamples of non-DTM cleavable linkers; sodium dithionite, Pd(0) and 340nm light, respectively, are shown as examples of means of cleavage;ATTO647N, Rox, Alexa488, BodipyFL and Cy5 are used as examples offluorophores; biotin or TCO are provided as examples of the anchors; andstreptavidin or tetrazine are used as examples of the anchor bindingmolecules. However, a variety of other cleavable groups in the linker,cleavage agents, fluorophores, anchors (e.g., DBCO, N₃, tetrazine), andanchor binding molecules (e.g., N₃, DBCO, TCO) are also feasible.

The first four schemes (A-D) are two color SBS schemes that require twofluorescence detection steps; schemes E-H are single color fluorescenceschemes that require three fluorescence detection steps to determine theincorporated nucleotide. Optional confirmatory imaging steps areincluded in some of these schemes. As with all the previously describedschemes, chasing is performed after adding the nucleotide analogues toguarantee that every primer has been extended so as to avoidasynchronous reactions, and washing is required between every step toremove the previous set of reagents and/or released dyes.

Scheme A: Two Color SBS: Imaging after Incorporation and Labeling (FIG.77).

In Scheme A, a set of nucleotide analogues, one with Anchor1 attached tothe 3′ position of the sugar via a dithiomethyl (DTM) cleavable linker,one with Anchor2 attached to the 3′ position via a DTM cleavable linker,one with Dye1 attached to the base via a DTM linker, and one with Dye2attached to the base via a DTM linker, is used. Imaging after additionof the four nucleotide analogues will indicate incorporation by eitherof two nucleotide analogues specifically. After labeling with both Dye1labeled Anchor1 binding molecule and Dye2 labeled Anchor2 bindingmolecule, imaging will reveal specifically incorporation by one of theother two nucleotide analogues. Cleavage with THP will then remove theremaining dyes and restore the 3′-OH group for subsequent sequencingcycles. In the example shown, Dye1 is Alexa488, Dye2 is Rox, Anchor1 isbiotin, and Anchor2 is TCO. Other combinations of dyes and anchors canalso be used.

Scheme B: Two Color SBS: Imaging after Incorporation and Cleavage (FIG.79).

In Scheme B, a set of nucleotide analogues, one with Dye1 attached tothe 3′ position of the sugar via a DTM cleavable linker, one with Dye2attached to the base via a DTM linker, one with Dye1 attached to thebase via an Azo cleavable linker, and one with Dye2 attached to the basevia an Azo cleavable linker, is used. In contrast to some previousschemes, here three of the nucleotide analogues have dyes on the basewith only one nucleotide analogue having a dye at the 3′ position. Afterincorporation, a Dye1 signal will indicate incorporation by either oftwo of the nucleotide analogues, while a Dye2 signal will indicateincorporation by either of the other two nucleotide analogues. Next,specific cleavage of the Azo linkers will reveal specifically which ofeither pair was incorporated. Cleavage with THP will then remove theremaining dyes and restore the 3′-OH group for subsequent sequencingcycles. In the example shown, Dye1 is Rox and Dye2 is BodipyFL. Otherdyes can also be used.

Scheme C: Two Color SBS with Dye Clusters: Imaging after Incorporationand Labeling (FIG. 81).

In Scheme C, a different set of nucleotide analogues is used: one with aDTM cleavable linker at the 3′ position attached to Anchor1, one with aDTM cleavable linker at the 3′ position attached to Anchor2, one with aDye1 cluster attached to the base via a DTM cleavable linker, and onewith a Dye2 cluster attached to the base via a DTM cleavable linker.Imaging immediately after incorporation will reveal specifically two ofthe nucleotide analogues. Next, labeling is carried out with an Anchor1binding molecule attached to a Dye1 cluster and an Anchor2 bindingmolecule attached to a Dye2 cluster. Appearance of Dye1 or Dye2fluorescence after this step will specifically identify incorporation bythe remaining two nucleotide analogues. The use of dye clustersconsisting of multiple dyes, such as 4, 5 or potentially 8 or moreidentical fluorophores, arranged so as to avoid quenching, will enableboth ensemble and single molecule sequencing. Cleavage with THP willthen remove the remaining dyes and restore the 3′-OH group forsubsequent sequencing cycles. In the example shown, the Dye1 cluster isa Rox cluster, the Dye2 cluster is an Alexa488 cluster. Anchor1 is TCO,and Anchor2 is biotin. Other combinations of dyes and anchors can alsobe used.

Scheme D: Two Color SBS with Energy Transfer Dyes: Imaging afterIncorporation and Labeling (FIG. 83).

In Scheme D, the set of nucleotide analogues consists of one with a DTMcleavable linker at the 3′ position attached to Anchor1, one with a DTMcleavable linker at the 3′ position attached to Anchor2, one afluorophore attached to the base via an DTM cleavable linker, and onewith a donor plus acceptor energy transfer pair of fluorophores attachedto the base via a DTM cleavable linker. Excitation of the donor dyeafter incorporation will reveal specifically two of the bases, one ifdonor emission is detected and the other if acceptor emissionpredominates. Next, labeling is carried out with Anchor1 bindingmolecule attached to the donor dye, and Anchor2 binding moleculeattached to the energy transfer dye pair cassette. Illumination at thedonor absorbance wavelength will reveal specifically incorporation ofthe remaining two nucleotide analogues. Cleavage with THP will thenremove the remaining dyes and restore the 3′-OH group for subsequentsequencing cycles. In the example shown, Anchor1 is biotin. Anchor2 isTCO, Dye1 is Rox, and the dye pair cassette has Rox as the donor and Cy5as the acceptor. Other combinations of dyes and anchors can also beused.

Scheme E: One Color SBS: Imaging after 2 Labeling Steps and Cleavage(FIG. 85).

In Scheme E, a set of nucleotide analogues with orthogonal cleavablelinker-anchor combinations is used: one with Anchor1 attached to the 3′position via a DTM cleavable linker, one with Anchor2 attached to the 3′position via a DTM cleavable linker, one with Anchor1 attached to thebase via an Azo cleavable linker, and one with Anchor2 attached to thebase via an Azo cleavable linker. After labeling with Anchor1 bindingmolecule attached to Dye1, imaging reveals two possible incorporatedbases. Next, after labeling with Anchor2 binding molecule attached toDye1, imaging confirms incorporation of the other two possibleincorporated bases. Finally, treatment with sodium dithionite willcleave the Azo linkers, specifically revealing which nucleotide wasadded. Cleavage with THP will then remove the remaining dyes and restorethe 3′-OH group for subsequent sequencing cycles. In the example shown,Dye1 is ATTO647N, Anchor1 is TCO, and Anchor2 is biotin. Othercombinations of dyes and anchors can also be used.

Scheme F: One Color SBS: Imaging after Incorporation, Labeling andCleavage (FIG. 87).

In Scheme F, the set of nucleotide analogues consists of one with Dye1attached to the 3′ position via a DTM cleavable linker, one with Anchor1attached to the 3′ position via a DTM cleavable linker, one with Dye1attached to the base via an Azo cleavable linker, and one with Anchor1attached to the base via an Azo cleavable linker. Dye1 detection afterincorporation reveals either of two of the possible incorporatednucleotide analogues. After labeling with Anchor1 binding moleculesattached to Dye1, imaging confirms incorporation of either of the othertwo possible nucleotide analogues. Finally, treatment with sodiumdithionite will cleave the Azo linkers, specifically revealing whichnucleotide was added. Cleavage with THP will then remove the remainingdyes and restore the 3′-OH group for subsequent sequencing cycles. Inthe example shown, Dye1 is Rox and Anchor1 is TCO. Other dyes andanchors can also be used.

Scheme G: One Color SBS: Imaging after Labeling and 2 Cleavage Steps(FIG. 89).

In Scheme G, three of the nucleotide analogues have the same Anchor1molecule attached to the 3′ position via either of three differentcleavable linkers (Allyl, 2-nitrobenzyl and DTM); the fourth nucleotidehas Dye1 attached to the base via a DTM linker. After incorporation,imaging reveals specifically one of the nucleotide analogues. Imagingfollowing labeling with the Anchor1 binding molecule attached to Dye1indicates the possibility of any of the remaining three nucleotideanalogues having been incorporated. Next, specific cleavage reactionsare carried out, one by one, with Pd(O) to cleave the allyl linkage,with 340 nm light to cleave the 2-nitrobenzyl linkage, and finally withTHP to cleave the DTM linkage, remove the remaining dye and restore the3′-OH group for subsequent sequencing cycles. Loss of fluorescence atany one of these steps will reveal specifically which nucleotide wasincorporated. In the example shown, Dye1 is ATTO0647N and Anchor1 isbiotin. Other dyes and anchors can also be used.

Scheme H: One Color SBS: Imaging after Incorporation, Labeling andCleavage (FIG. 91).

In Scheme H, a set of nucleotide analogues with orthogonal cleavablelinker-anchor-dye cluster combinations is used: one with a Dye1 clusterattached to the base via a DTM cleavable linker, one with a Dye1 clusterattached to the base via an Azo cleavable linker, one with Anchor1attached to the 3′ position via a DTM cleavable linker, and one withAnchor2 attached to the 3′ position via a DTM cleavable linker. Afterincorporation, fluorescence indicates the possibility of either of twonucleotide analogues. Imaging after labeling with a Dye1 clusterattached to an Anchor1 binding molecule will specifically reveal one ofthe remaining two nucleotide analogues, and imaging after subsequentlabeling with a Dye1 cluster attached to Anchor2 binding molecule willspecifically reveal the other of the remaining two nucleotide analogues.Imaging following cleavage with sodium dithionite to remove dyes withAzo linkers will specifically reveal either of the first two nucleotideanalogues having been incorporated. Finally, cleavage with THP willremove the remaining dyes and restore the 3′-OH group for subsequentsequencing cycles. As with Scheme C, the use of dye clusters withmultiple dyes, such as of 4, 5 or potentially 8 or more identicalfluorophores will enable ensemble and single molecule sequencing. In theexample shown, the Dye1 cluster is a Rox cluster, Anchor1 is biotin, andAnchor2 is TCO. Other combinations of dyes and anchors can also be used.

What is claimed is:
 1. A method of incorporating a nucleotide analogueinto a nucleic acid sequence comprising combining a thermophilic nucleicacid polymerase, a primer hybridized to nucleic acid template, and anucleotide analogue comprising a detectable label, within a reactionvessel and allowing said thermophilic nucleic acid polymerase toincorporate said nucleotide analogue into said primer therebyincorporating a nucleotide analogue into a nucleic acid sequence
 2. Themethod of claim 1, wherein said nucleotide analogue has the formula:

wherein the symbol “----” is a non-covalent bond; B is a base oranalogue thereof; L¹ is covalent linker; L² is covalent linker; L⁴ iscovalent linker; X is a bond, O, NR^(6A), or S; R³ is triphosphate orhigher polyphosphate; R^(4A) is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R^(6A) is hydrogen, —OH, —CF₃,—CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br,—CH₂I, —CN, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁵ is adetectable label, anchor moiety, or affinity anchor moiety; R⁶ is —CF₃,—CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br,—CH₂I, —CN, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen or —OR^(7A), wherein R^(7A) is hydrogen or apolymerase-compatible moiety; R¹² is a complementary affinity anchormoiety binder; and R¹³ is a detectable label. 3-4. (canceled)
 5. Themethod of claim 2, wherein L¹ is a substituted or unsubstitutedmethylene, wherein L¹ is substituted with a substituted or unsubstitutedC₁-C₆ alkylene, substituted or unsubstituted 2 to 6 memberedheteroalkylene, substituted or unsubstituted C₃-C₆ cycloalkylene,substituted or unsubstituted 3 to 6 membered heterocycloalkylene,substituted or unsubstituted phenyl, or substituted or unsubstituted 5to 6 membered heteroarylene. 6-12. (canceled)
 13. The method of claim 2,wherein L² is a cleavable linker comprising a dialkylketal linker, anazo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl linker, or anitrobenzyl linker.
 14. The method of claim 2, wherein: L² isL^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A), L^(2B), L^(2C), L^(2D),and L^(2E) are independently a bond, —NN—, —NHC (O)—, —C(O) NH—,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene; wherein at leastone of L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) is not a bond. 15-22.(canceled)
 23. The method of claim 2, wherein L² is a substituted orunsubstituted 4 to 10 membered heteroalkylene.
 24. (canceled)
 25. Themethod of claim 2, wherein L² is —C(CH₃)₂CH₂NHC(O)—,


26. (canceled)
 27. (canceled)
 28. The method of claim 2, wherein R^(4A)is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂,—CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted or unsubstituted alkyl,or substituted or unsubstituted heteroalkyl. 29-32. (canceled)
 33. Themethod of claim 2, wherein R^(6A) is —OH, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl. 34-38. (canceled)
 39. The method of claim 2, wherein R⁶ is—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl,—CH₂Br, —CH₂I, —CN, substituted or unsubstituted alkyl, or substitutedor unsubstituted heteroalkyl. 40-43. (canceled)
 44. The method of claim2, wherein R⁵ is

unsubstituted ethynyl,

45-47. (canceled)
 48. The method of claim 2, wherein R⁷ is hydrogen.49-52. (canceled)
 53. The method of claim 2, wherein R¹² is astreptavidin moiety. 54-58. (canceled)
 59. The method of claim 2,wherein L⁴ is a bond, substituted or unsubstituted alkylene, substitutedor unsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene. 60-61. (canceled)
 62. The method of claim 2, whereinR¹²-L⁴-R¹³ has the formula:


63. (canceled)
 64. (canceled)
 65. The method of claim 2, wherein R¹² isselected from the group consisting of:

a streptavidin moiety,

unsubstituted ethynyl,


66. The method of claim 1, wherein the nucleotide analogue has theformula:


67. A thermophilic nucleic acid polymerase complex, wherein saidthermophilic nucleic acid polymerase is bound to a nucleotide analoguehas the formula:

wherein: the symbol “----” is a non-covalent bond; B is a base oranalogue thereof; L¹ is covalent linker; L² is covalent linker; L⁴ iscovalent linker; R³ is monophosphate, triphosphate or higherpolyphosphate, or a nucleic acid; R⁴ is hydrogen, —CF₃, —CCl₃, —CBr₃,—CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R^(4B) is hydrogen, —CF₃,—CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br,—CH₂I, —CN, —X—R′, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; X is abond, O, NR⁶, or S; R^(6A) is —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂,—CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁵ is a detectable label, anchor moiety, oraffinity anchor moiety; R⁶ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁷ is hydrogen or —OR^(7A), wherein R^(7A) ishydrogen or a polymerase-compatible moiety; R¹² is a complementaryaffinity anchor moiety binder; and R¹³ is a detectable label. 68.(canceled)
 69. A method for sequencing a nucleic acid, comprising: (i)incorporating in series with a thermophilic nucleic acid polymerase,within a reaction vessel, one of four different labeled nucleotideanalogues into a primer to create an extension strand, wherein saidprimer is hybridized to said nucleic acid and wherein each of the fourdifferent labeled nucleotide analogues comprise a unique detectablelabel; (ii) detecting said unique detectable label of each incorporatednucleotide analogue, so as to thereby identify each incorporatednucleotide analogue in said extension strand, thereby sequencing thenucleic acid; wherein each of said four different labeled nucleotideanalogues are of the structure formula:

wherein in the first of said four different labeled nucleotideanalogues, B is a thymine or uracil hybridizing base; in the second ofsaid four different labeled nucleotide analogues, B is an adeninehybridizing base; in the third of said four different labeled nucleotideanalogues, B is a guanine hybridizing base; and in the fourth of saidfour different labeled nucleotide analogues, B is a cytosine hybridizingbase; the symbol “----” is a non-covalent bond; B is a base or analoguethereof; L¹ is covalent linker; L² is covalent linker; L⁴ is covalentlinker; X is a bond, O, NR^(6A), or S; R³ is a triphosphate or higherpolypohsphate; R^(4A) is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁵ is a detectable label, anchor moiety, oraffinity anchor moiety; R⁶ is —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CHCl₂,—CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —N, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(6A) is —OH, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CHCl₂, —CHBr₂, —CHI₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, —CN,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁷ is hydrogen or —OR^(7A),wherein R^(7A) is hydrogen or a polymerase-compatible moiety; R¹² is acomplementary affinity anchor moiety binder; and R¹³ is a detectablelabel. 70-71. (canceled)